Histone deacetylases, and uses related thereto

ABSTRACT

The present invention concerns the discovery that proteins encoded by a family of genes, termed here HDx-related genes, which are involved in the control of chromatin structure and, thus in transcription and translation. The present invention makes available compositions and methods that can be utilized, for example to control cell proliferation and differentiation in vitro and in vivo.

GOVERNMENT FUNDING

Work described herein was supported in part by funding from the NationalInstitute of Health. The United States Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The organization of regulatory DNA elements into precise chromatinstructures is important for both DNA replication and transcription invivo (Lee et al. (1993) Cell 72:73-84; Felsenfeld (1992) Nature.355:219). In eukaryotic cells, nuclear DNA exists as a hierarchy ofchromatin structures, resulting in the compaction of nuclear DNA about10,000 fold (Davie and Hendzel (1994) J. Cell. Biochem. 55:98). Therepeating structural unit in the extended 10 nm fibre form of chromatinis the nucleosome (van Holde (1988) Chromatin. New York:Springer-Verlag). The nucleosome consists of 146 bp of DNA wrappedaround a protein core of the histones H2A, H2B, H3, and H4, known as thecore histones. These histones are arranged as an (H3-H4)₂ tetramer andtwo H2A-H2B dimers positioned on each face of the tetramer. The DNAjoining the nucleosomes is called linker DNA; it is to the linker DNA towhich the H1 or linker histones bind. The 10 nm fibre is compactedfurther into the 30 nm fibre. Linker histones and amino-terminal regions(“tails”) of the core histones maintain the higher order folding ofchromatin (Garcia Ramirez et al. (1992) J. Biol Chem 267:19587). Thischromatin structure must be relaxed when DNA is transcribed ortranslated.

Histones of the nucleosome core particle are subject to reversibleacetylation at the ε-amino group of lysines present in their aminoterminus (Csordas et al. (1990) Biochem J 265:23-38). Transcriptionallysilent regions of the genome are enriched in underacetylated histone H4(Turner (1993) Cell 75:5-8), and histone hyperacetylation facilitatesthe ability of transcription factor TFIIIA to bind to chromatintemplates (Lee et al. (1993) Cell 72:73-84). Recent genetic, biochemicaland immunological approaches have provided substantial evidenceindicating that histones associated with actively transcribed genes aremore highly acetylated than those from nontranscribed regions. While notwishing to be bound by any particular theory, histone acetylation mayinfluence transcription at several stages, for example, by causingtranscription factors to bind or by inducing structural transitions inchromatin, or by facilitating histone displacement and repositioningduring polymerase elongation.

The acetylation and deacetylation are catalyzed by specific enzymes,histone acetyltransferase and deacetylase, respectively, and the netlevel of the acetylation is controlled by the equilibrium between theseenzymes. The steady state level of acetylation and the rates at whichacetate groups are turned over vary both between and within differentcell types, with half-lives that vary from a few minutes to severalhours. Although a histone acetyltransferase gene (HAT1) has beenidentified in yeast (Kelff et al. (1995) J. Biol. Chem.270:24674-24677), the molecular entities responsible for histonedeacetylation were heretofore unknown in the art.

The identification of the mechanism by which histones are deacetylatedwould be of great benefit in the control of gene transcription and thecell cycle.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a novel family ofgenes, and gene products, expressed in mammals, which genes are referredto hereinafter as the “histone deacetylase” genes or “HDx” gene family,the products of which are referred to as histone deacetylases or HDxproteins.

In general, the invention features isolated HDx polypeptides, preferablysubstantially pure preparations of one or more of the subject HDxpolypeptides. The invention also provides recombinantly produced HDxpolypeptides. In preferred embodiments the polypeptide has a biologicalactivity including an ability to deacetylate an acetylated histonesubstrate, preferably a substrate analog of histone H3 and/or H4. Inother embodiments the HDx polypeptides of the present invention bind totrapoxin or to trichostatin, such binding resulting in the inhibition adeacetylase activity of the HDx polypeptide. However, HDx polypeptideswhich specifically antagonize such activities, such as may be providedby dominant negative mutants, are also specifically contemplated.

The HDx polypeptides disclosed herein are capable of modulatingproliferation, survival and/or differentiation of cells, because oftheir ability to alter chromatin structure by deacetylating histonessuch as H3 or H4. Moreover, in preferred embodiments, the subject HDxproteins have the ability to modulate cell growth by influencing cellcycle progression or to modulate gene transcription.

In one embodiment, the polypeptide is identical with or homologous to anHDx protein. Exemplary HDx polypeptide include amino acid sequencesrepresented in any one of SEQ ID Nos 5-8. Related members of the HDxfamily are also contemplated, for instance, an HDx polypeptidepreferably has an amino acid sequence at least 85% homologous to apolypeptide represented by one or more of the polypeptides designatedSEQ ID Nos: 5-8, though polypeptides with higher sequence homologies of,for example, 88, 90% and 95% or are also contemplated. In oneembodiment, the HDx polypeptide is encoded by a nucleic acid whichhybridizes under stringent conditions with a nucleic acid sequencerepresented in one or more of SEQ ID Nos. 14. Homologs of the subjectHDx proteins also include versions of the protein which are resistant topost-translation modification, as for example, due to mutations whichalter modification sites (such as tyrosine, threonine, serine oraspargine residues), or which inactivate an enzymatic activityassociated with the protein.

The HDx polypeptide can comprise a full length protein, such asrepresented in SEQ ID No. 5, or it can comprise a fragment correspondingto particular motifs/domains, or to arbitrary sizes, e. g., at least 5,10, 25, 50, 100, 150 or 200 amino acids in length. In preferredembodiments, the polypeptide, or fragment thereof, specificallydeacetylates histone H4. In other preferred embodiments, the HDxpolypeptide includes both a ν motif (SEQ ID No. 12) and a χ motif (SEQID No. 14), preferably a ν motif represented in the general formula SEQID No. 13, and a χ motif represented in the general formula SEQ ID No.15.

In certain preferred embodiments, the invention features a purified orrecombinant HDx polypeptide having a molecular weight in the range of 40kd to 60 kd. For instance, preferred HDx polypeptides, have molecularweights in the range of 50 kd to about 60 kd, even more preferably inthe range of 53-58 kd. It will be understood that certainpost-translational modifications, e. g., phosphorylation, prenylationand the like, can increase the apparent molecular weight of the HDxprotein relative to the unmodified polypeptide chain.

The subject proteins can also be provided as chimeric molecules, such asin the form of fusion proteins. For instance, the HDx protein can beprovided as a recombinant fusion protein which includes a secondpolypeptide portion, e. g., a second polypeptide having an amino acidsequence unrelated (heterologous) to the HDx polypeptide, e. g. thesecond polypeptide portion is glutathione-S-transferase, e. g. thesecond polypeptide portion is an enzymatic activity such as alkalinephosphatase, e. g. the second polypeptide portion is an epitope tag.

In yet another embodiment, the invention features a nucleic acidencoding a an HDx polypeptide, or polypeptide homologous thereto, whichpolypeptide has the ability to modulate, e. g., either mimic orantagonize, at least a portion of the activity of a wild-type HDxpolypeptide. Exemplary HDx-encoding nucleic acid sequences arerepresented by SEQ ID Nos: 1-4.

In another embodiment, the nucleic acid of the present inventionincludes a coding sequence which hybridizes under stringent conditionswith one or more of the nucleic acid sequences in SEQ ID Nos: 1-4. Thecoding sequence of the nucleic acid can comprise a sequence which isidentical to a coding sequence represented in one of SEQ ID Nos: 1-4, orit can merely be homologous to one or more of those sequences. Inpreferred embodiments, the nucleic acid encodes a polypeptide whichspecifically modulates, by acting as either an agonist or antagonist,the enzymatic activity of an HDx polypeptide.

Furthermore, in certain preferred embodiments, the subject HDx nucleicacid will include a transcriptional regulatory sequence, e. g. at leastone of a transcriptional promoter or transcriptional enhancer sequence,which regulatory sequence is operably linked to the HDx gene sequence.Such regulatory sequences can be used in to render the HDx gene sequencesuitable for use as an expression vector. This invention alsocontemplates the cells transfected with said expression vector whetherprokaryotic or eukaryotic and a method for producing HDx proteins byemploying said expression vectors.

In yet another embodiment, the nucleic acid hybridizes under stringentconditions to a nucleic acid probe corresponding to at least 12consecutive nucleotides of either sense or antisense sequence of one ormore of SEQ ID Nos: 1-4; though preferably to at least 25 consecutivenucleotides; and more preferably to at least 40, 50 or 75 consecutivenucleotides of either sense or antisense sequence of one or more of SEQID Nos: 14.

Yet another aspect of the present invention concerns an immunogencomprising an HDx polypeptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for anHDx polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprising an antigenic determinant, e.g. a unique determinant, from aprotein represented by one of SEQ ID Nos. 5-8.

A still further aspect of the present invention features antibodies andantibody preparations specifically reactive with an epitope of the HDximmunogen.

The invention also features transgenic non-human animals, e.g. mice,rats, rabbits, chickens, frogs or pigs, having a transgene, e.g.,animals which include (and preferably express) a heterologous form of anHDx gene described herein, or which misexpress an endogenous HDx gene,e.g., an animal in which expression of one or more of the subject HDxproteins is disrupted. Such a transgenic animal can serve as an animalmodel for studying cellular and tissue disorders comprising mutated ormis-expressed HDx alleles or for use in drug screening.

The invention also provides a probe/primer comprising a substantiallypurified oligonucleotide, wherein the oligonucleotide comprises a regionof nucleotide sequence which hybridizes under stringent conditions to atleast 12 consecutive nucleotides of sense or antisense sequence of SEQID Nos: 1-4, or naturally occurring mutants thereof. Nucleic acid probeswhich are specific for each of the HDx proteins are contemplated by thepresent invention, e.g. probes which can discern between nucleic acidencoding a human or bovine HD. In preferred embodiments, theprobe/primer further includes a label group attached thereto and able tobe detected. The label group can be selected, e.g., from a groupconsisting of radioisotopes, fluorescent compounds, enzymes, and enzymeco-factors. Probes of the invention can be used as a part of adiagnostic test kit for identifying dysfunctions associated withmis-expression of an HDx protein, such as for detecting in a sample ofcells isolated from a patient, a level of a nucleic acid encoding asubject HDx protein; e.g. measuring an HDx mRNA level in a cell, ordetermining whether a genomic HDx gene has been mutated or deleted.These so called “probes/primers” of the invention can also be used as apart of “antisense” therapy which refers to administration or in situgeneration of oligonucleotide probes or their derivatives whichspecifically hybridize (e.g. bind) under cellular conditions, with thecellular mRNA and/or genomic DNA encoding one or more of the subject HDxproteins so as to inhibit expression of that protein, e.g. by inhibitingtranscription and/or translation. Preferably, the oligonucleotide is atleast 12 nucleotides in length, though primers of 25, 40, 50, or 75nucleotides in length are also contemplated.

In yet another aspect, the invention provides an assay for screeningtest compounds for inhibitors, or alternatively, potentiators, of aninteraction between an HDx protein and an HDx binding protein or nucleicacid sequence. An exemplary method includes the steps of (i) combiningan HDx polypeptide or fragment thereof, an HDx target polypeptide (suchas a histone or RpAp48), and a test compound, e.g., under conditionswherein, but for the test compound, the HDx protein and targetpolypeptide are able to interact; and (ii) detecting the formation of acomplex which includes the HDx protein and the target polypeptide eitherby directly quantitating the complex, the deacetylase activity of theHDx protein, or by measuring inductive effects of the HDx protein. Astatistically significant change, such as a decrease, in the formationof the complex in the presence of a test compound (relative to what. isseen in the absence of the test compound) is indicative of a modulation,e.g., inhibition, of the interaction between the HDx protein and itstarget polypeptide.

Furthermore, the present invention contemplates the use of otherhomologs of the HDx polypeptides or bioactive fragments thereof togenerate similar assay formats. In one embodiment, the drug screeningassay can be derived with a fungal homolog of an HDx protein, such asRPD3, in order to identify agents which inhibit histone deacetylation ina yeast cell.

Yet another aspect of the present invention concerns a method formodulating one or more of growth, differentiation, or survival of amammalian cell by modulating HDx bioactivity, e.g., by inhibiting thedeacetylase activity of HDx proteins, or disrupting certainprotein-protein interactions. In general, whether carried out in vivo,in vitro, or in situ, the method comprises treating the cell with aneffective amount of an HDx therapeutic so as to alter, relative to thecell in the absence of treatment, at least one of (i) rate of growth,(ii) differentiation, or (iii) survival of the cell. Accordingly, themethod can be carried out with HDx therapeutics such as peptide andpeptidomimetics or other molecules identified in the above-referenceddrug screens which antagonize the effects of a naturally-occurring HDxprotein on said cell. Other HDx therapeutics include antisenseconstructs for inhibiting expression of HDx proteins, and dominantnegative mutants of HDx proteins which competitively inhibitprotein-substrate and/or protein-protein interactions upstream anddownstream of the wild-type HDx protein.

In an exemplary embodiment the subject method is used to treat tumorcells by antagonizing HDx activity and blocking cell cycle progression.In one embodiment, the subject method includes the treatment oftesticular cells, so as modulate spermatogenesis. In another embodiment,the subject method is used to modulate osteogenesis, comprising thetreatment of osteogenic cells with an HDx polypeptide. Likewise, wherethe treated cell is a chondrogenic cell, the present method is used tomodulate chondrogenesis. In still another embodiment, HDx polypeptidescan be used to modulate the differentiation of progenitor cells, e.g.,the method can be used to cause differentiation of a hematopoieticcells, neuronal cells, or other stem/progenitor cell populations, tomaintain a that cell in a differentiated state, and/or to enhance thesurvival of a differentiated cell, e.g., to prevent apoptosis or otherforms of cell death.

In addition to such HDx therapeutic uses, anti-fungal agents developedwith such screening assays as described herein can be used, for example,as preservatives in foodstuff, feed supplement for promoting weight gainin livestock, or in disinfectant formulations for treatment ofnon-living matter, e.g., for decontaminating hospital equipment androoms. In similar fashion, assays provided herein will permit selectionof deacetylase inhibitors which discriminate between the human andinsect deacetylase enzymes. Accordingly, the present invention expresslycontemplates the use and formulations of the deacetylase inhibitors ininsecticides, such as for use in management of insects like the fruitfly. Moreover, certain of the inhibitors can be selected on the basis ofinhibitory specificity for plant HDx-related activities relative to themammalian enzymes. Thus, the present invention specifically contemplatesformulations of deacetylase inhibitors for agricultural applications,such as in the form of a defoliant or the like.

The present method is applicable, for example, to cell culturetechnique, such as in the culturing of hematopoietic cells and othercells whose survival or differentiative state is dependent on HDxfunction. Moreover, HDx agonists and antagonists can be used fortherapeutic intervention, such as to enhance survival and maintenance ofcells, as well as to influence organogenic pathways, such as tissuepatterning and other differentiation processes. In an exemplaryembodiment, the method is practiced for modulating, in an animal, cellgrowth, cell differentiation or cell survival, and comprisesadministering a therapeutically effective amount of an HDx polypeptideto alter, relative the absence of HDx treatment, at least one of (i)rate of growth, (ii) differentiation, or (iii) survival of one or morecell-types in the animal.

Another aspect of the present invention provides a method of determiningif a subject, e.g. a human patient, is at risk for a disordercharacterized by unwanted cell proliferation or aberrant control ofdifferentiation. The method includes detecting, in a tissue of thesubject, the presence or absence of a genetic lesion characterized by atleast one of (i) a mutation of a gene encoding an HDx protein, e.g.represented in one of SEQ ID Nos: 1-4, or a homolog thereof; or (ii) themis-expression of an HDx gene. In preferred embodiments, detecting thegenetic lesion includes ascertaining the existence of at least one of: adeletion of one or more nucleotides from an HDx gene; an addition of oneor more nucleotides to the gene, a substitution of one or morenucleotides of the gene, a gross chromosomal rearrangement of the gene;an alteration in the level of a messenger RNA transcript of the gene;the presence of a non-wild type splicing pattern of a messenger RNAtranscript of the gene; or a non-wild type level of the protein.

For example, detecting the genetic lesion can include (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequence ofan HDx gene, e.g. a nucleic acid represented in one of SEQ ID Nos: 1-4,or naturally occurring mutants thereof, or 5′ or 3′ flanking sequencesnaturally associated with the HDx gene; (ii) exposing the probe/primerto nucleic acid of the tissue; and (iii) detecting, by hybridization ofthe probe/primer to the nucleic acid, the presence or absence of thegenetic lesion; e.g. wherein detecting the lesion comprises utilizingthe probe/primer to determine the nucleotide sequence of the HDx geneand, optionally, of the flanking nucleic acid sequences. For instance,the probe/primer can be employed in a polymerase chain reaction (PCR) orin a ligation chain reaction (LCR). In alternate embodiments, the levelof an HDx protein is detected in an immunoassay using an antibody whichis specifically immunoreactive with the HDx protein.

In another aspect, the invention provides compounds useful forinhibition of HDxs. In a preferred embodiment, an HDx inhibitor compoundof the invention can be represented by the formula A-B-C, in which A isa specificity element for selective binding to an HDx, B is a linkerelement, and C is an electrophilic moiety capable of reacting with anucleophilic moiety of an HDx; with the proviso that the compound is notbutyrate, trapoxin, or trichostatin.

For instance, in one embodiment, there is provided a composition forinhibiting a histone deacetylase comprising a compound represented bythe general formula A-B-C, wherein

A is selected from the group consisting of cycloalkyls, unsubstitutedand substituted aryls, heterocyclyls, amino acyls, and cyclopeptides;

B is selected from the group consisting of substituted and unsubstitutedC₄-C₈ alkylidenes, C₄-C₈ alkenylidenes, C₄-C₈ alkynylidenes, and—D—E—F)—, in which D and F are, independently, absent or represent aC₂-C₇ alkylidene, a C₂-C₇ alkenylidene or a C₂-C₇ alkynylidene, and Erepresents O, S, or NR′, in which R′ represents H, a lower alkyl, alower alkenyl, a lower alkynyl, an aralkyl, aryl, or a heterocyclyl; and

C is selected from the group consisting of

and a boronic acid; in which Z represents O, S, or NR₅, and Y; R₅represents a hydrogen, an alkyl, an alkoxycarbonyl, an aryloxycarbonyl,an alkylsulfonyl, an arylsulfonyl or an aryl; R′₆ represents hydrogen,an alkyl, an alkenyl, an alkynyl or an aryl; and R₇ represents ahydrogen, an alkyl, an aryl, an alkoxy, an aryloxy, an amino, ahydroxylamino, an alkoxylamino or a halogen; with the proviso that thecompound is not trapoxin.

In another preferred embodiment, the compound represented by the generalformula A-B-C, wherein

A is selected from the group consisting of cycloalkyls, unsubstitutedand substituted aryls, heterocyclyls, amino acyls, and cyclopeptides;

B is selected from the group consisting of substituted and unsubstitutedC₄-C₈ alkylidenes, C₄-C₈ alkenylidenes, C₄-C₈ alkynylidenes, and—(D—E—F)—, in which D and F are, independently, absent or representC₂-C₇ alkylidenes, C₂-C₇ alkenylidenes or C₂-C₇ alkynylidenes, and Erepresents O, S, or NR′, in which R′ represents H, a lower alkyl, alower alkenyl, a lower alkynyl, an aralkyl, an aryl, or a heterocyclyl;and

C is selected from the group consisting of

in which R₉ represents a hydrogen, an alkyl, an aryl, a hydroxyl, analkoxy, an aryloxy or an amino, with the proviso that the inhibitorcompound is not trichostatin.

In still another preferred embodiment, the compound is represented bythe general formula A-B-C, wherein

A is selected from the group consisting of cycloalkyls, unsubstitutedand substituted aryls, heterocyclyls, amino acyls, and cyclopeptides;

B is selected from the group consisting of substituted and unsubstitutedC₄-C₈ alkylidenes, C₄-C₈ alkenylidenes, C₄-C₈ alkynylidenes, and—(D—E—F)—, in which D and F are, independently, absent or a C₂-C₇alkylidene, a C₂-C₇ alkenylidene, or a C₂-C₇ alkynylidene, and Erepresents O, S, or NR′, in which R′ is H, lower alkyl, lower alkenyl,lower alkynyl, aralkyl, aryl, or heterocyclyl; and

C represents

in which Y is O or S, and R₇ represents a hydrogen, an alkyl, an aryl,an alkoxy, an aryloxy, an amino, a hydroxylamino, an alkoxylamino or ahalogen.

The present invention also contemplates pharmaceutical preparations ofsuch compounds, e.g., in an amount effective for inhibitingproliferation of a cell, formulated in a pharmaceutically acceptablediluent.

Moreover, such compounds can be used for modulating one or more ofgrowth, differentiation, or survival of a mammalian cell responsive toHDx-mediated histone deacetylation, by treating the cell with aneffective amount of the deacetylase inhibitor so as to modulate thedeacetylase activity and alter, relative to the cell in the absence ofthe agent, at least one of (i) the rate of growth, (ii) thedifferentiation state, or (iii) the rate of survival of the cell.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); TranscriptionAnd. Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology (AcademicPress, Inc., N.Y. ); Gene Transfer Vectors For Mammalian Cells (J. H.Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory);Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986);Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986).

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the chemical structures of trapoxin andtrichostatin, natural products that inhibit the enzymatic deacetylationof lysine residues near the NH₂-terminus of histones. The epoxyketoneside chain of trapoxin is approximately isosteric with N-acetyl lysineand likely alkylates an active site nucleophile.

FIG. 1B illustrates the copurification of trapoxin binding and histonedeacetylase activities. Nuclear proteins from bovine thymus wereprecipitated with ammonium sulfate and fractionated on a Mono Q column.Trapoxin binding was assayed by charcoal precipitation with[³H]trapoxin. For the histone deacetylase assay, a peptide correspondingto bovine histone H4 (1-24) was synthesized. The peptide was chemicallyacetylated with sodium [³H]acetate (5.3 Ci/mmol, New EnglandNuclear)/BOP reagent (Aldrich) and purified by reverse phase HPLC. Twomicroliters of [³H]peptide(˜40,000 dpm) were used per 200 μl assay.After incubation at 37° C. for one hour, the reaction was quenched with1 M HCl/0.16 M acetic acid (50 μl). Released [³H]acetic acid wasextracted with 600 μl of ethyl acetate and quantified by scintillationcounting. Pretreatment of crude or partially purified enzyme withtrapoxin or trichostatin (20 nM) for 30 min. at 4° C. abolisheddeacetylase activity. A₂₈₀=absorbance at 280 nm.

FIG. 2A shows the synthesis of K-trap and the K-trap affinity matrix.K-trap contains a protected lysine residue in place of the phenylalanineat position two in trapoxin. Alloc=allyloxycarbonyl.

FIG. 2B is a silver stained gel showing bovine and human trapoxinbinding proteins. Proteins bound to the K-trap affinity matrix in thepresence or absence of trapoxin or trichostatin were eluted by boilingin SDS loading buffer and analyzed by SDS-PAGE (9% gel). Nuclearproteins from human Jurkat T cells were prepared identically to thosefrom bovine thymus (FIG. 1B). Molecular size standards (in kilodaltons)are indicated to the right.

FIG. 3A is the predicted amino acid sequence of human HD1 (SEQ D No. 5).An in-frame stop codon was found upstream of the starting methionine.Regions equivalent to microsequenced tryptic peptides from the purifiedbovine protein are boxed. Underlined amino acids 319-334 and 467-482denote the sequences of synthetic peptides that were conjugated to KLHand used to generate polyclonal antisera. Abbreviations for the aminoacid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S,Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

FIG. 3B is a protein immunoblot analogous to the silver stained gel inFIG. 2B, showing the relationship between bovine p46-p49 and human p55(top panels) and confirming the identity of p50 (bovine and human) asRbAp48 (bottom panels). Proteins eluted from the K-trap affinity matrix(FIG. 2) were separated by SDS-PAGE and transferred to Immobilon-P(Millipore). Blots were probed with polyclonal anti-HDI (319-336) ormonoclonal anti-RbAp48 and bound antibodies were detected with enhancedchemiluminescence (Amersham).

FIG. 4A is an immunoprecipitation of endogenous histone deacetylaseactivity with affinity purified anti-HD1(467-482) antibodies.Anti-HD1(467-482) immunoprecipitates from equivalent amounts of Jurkatnuclear extract (1 mg nuclear protein supplemented with 0.5 M NaCl, 1%BSA, and 0.1% NP-40) were isolated in the presence or absence ofHD1(467-482) peptide competitor. After resuspending theimmunoprecipitates in HDx buffer [20 mM tris (pH 8), 150 mM NaCl, 10%glycerol], inhibitors were added as indicated, and histone deacetylaseactivity was measured as described in FIG. 1A.

FIG. 4B shows the coprecipitation of HD1 and RbAp48, as detected byprotein immunoblot analysis.

FIG. 4C demonstrates the histone deacetylase activity of recombinantHD1-F. Tag Jurkat cells (Clipstone et al. (1992) Nature 357, 695-7) weretransfected with pFJ5 (vector alone) or pBJ5/HD1-F (encodingCOOH-terminal FLAG epitope tagged HD1) by electroporation and detergentlysates were prepared [0.5% Triton X-100, 50 mM tris (pH 8), 100 mMNaCl, 10% glycerol]. Anti-FLAG antibodies conjugated to agarose beads(IBI) were used to immunoprecipitates recombinant HD1 in the presence orabsence of FLAG peptide competitor, and histone deacetylase activity wasmeasured as described above.

FIG. 4D shows the interaction between recombinant HD1-F and the K-trapaffinity matrix. Lysates from Jurkat cells transfected with pBJ5/HD1-Fwere incubated with the K-trap affinity matrix in the presence orabsence of inhibitors. Immunoblots of the eluted proteins were probedwith the anti-FLAG M2 monoclonal antibody (IBI).

FIGS. 5A and 5B are sequence alignments for various HDx and HDx-relatedcDNAs and proteins, respectively.

FIG. 6 depicts exemplary specificity elements (A), linker elements (B),and electrophilic moieties (C) for generating compounds which arecapable of reacting with a nucleophilic moiety of an HDx protein.

FIG. 7 illustrates an exemplary synthesis of trichostatin analogs.

FIGS. 8A-8C illustrate a synthesis of tritiated Trapoxin B.

FIGS. 9A-9C depict a synthesis of the K-trap and K-trap affinity matrix

DETAILED DESCRIPTION OF THE INVENTION

The positioning of nucleosomes relative to particular regulatoryelements in genomic DNA has emerged as a mechanism for managing theassociation of sequence-specific DNA-binding proteins with promoters,enhancers and other transcriptional regulatory sequences. Twomodifications to nucleosomes have been observed to influence theassociation of DNA-binding proteins with chromatin. Depletion ofhistones H2A/H2B from the nucleosome facilitates the binding of RNApolymerase II (Baer et al. (1983) Nature 301:482-488) and TFIIIA (Hayeset al. (1992) PNAS 89:1229-1233). Likewise, acetylation of the corehistones apparently destabilizes the nucleosome and is thought tomodulate the accessibility of transcription factors to their respectiveenhancer and promoter elements (Oliva et al. (1990) Nuc Acid Res18:2739-2747; and Walker et al. (1990) J Biol Chem 265:5622-5746). Inboth cases, overall histone-DNA contacts are altered.

In one aspect, the present invention concerns the discovery of a familyof genes in mammals, the gene products of which are referred to hereinas “histone deacetylases” or “HDx's”. Experimental evidence indicates afunctional role for the HDx gene products as catalysts of thedeacetylation of histones in mammalian cells, and accordingly play arole in determining tissue fate and maintenance. For instance, theresults provided below indicate that proteins encoded by the HDx genesmay participate, under various circumstances, in the control ofproliferation, differentiation and cell death.

The family of HDx gene apparently encode at least three differentsub-families, e.g., paralogs, and have been identified from the cells ofvarious mammals. The HDx proteins were first isolated from bovine thymusnuclei by use of a binding assay which exploited the ability oftrapoxin, an inhibitor of histone deacetylase activity, to isolateproteins which co-purified with a histone acetylase activity. Thepartial identity of the isolated proteins were determined by peptidemicrosequencing, and primers based on the peptide sequences were used toclone human cDNAs from a T cell library. One of the HDx gene productsdescribed below is referred to herein as HD1, and is represented in SEQID No. 1 (nucleotide) and SEQ ID No. 2 (amino acid).

A search of expressed sequence tag (EST) libraries turned up partialsequences for human HDx transcripts, and revealed the existence of atleast two other human HDx genes related to HD1, these other paralogsreferred to herein as HD2 and HD3. Nucleotide and amino acid sequencesfor partial clones of other human HDx homologs are provided by SEQ IDNos. 2-4 and 6-8, respectively.

Analysis of the HDx sequences indicated no obvious similarities with anypreviously identified domains or motifs. However, the fact that eachfull-length clone lacks a signal sequence, along with the observationthat proteins can be detected in the nucleus, indicates that the HDxgenes encode intracellular proteins.

Careful inspection of the HDx clones suggests at least two novel motifs,one or both of which may be characteristic of at least subfamilies ofthe mammalian HDx family. The first apparently conserved structuralelement of the HDx family occurs in the N-terminal portion of themolecule, and is designated herein as the “ν motif”. With reference tohuman HD1, the ν motif corresponds to amino acid residues Asp130-Phe198. By alignment of the HDx sequences, the element is represented bythe consensus sequence:

DXXXNXXGGLHHAKKXEASGFCYXNDIVXXIXELLXYHXRVXYIDXDXHHGHGXEAFYXTDRVMTXSF,  (SEQID No. 12)

more preferably by the consensus sequence:

DIAX1NWAGGLHHAKKX2EASGFCYVNDIVX3X4ILELLKYHX5RVLYIDIDIHHGDGX6EAFYX7TDRVMTVSF  (SEQID No. 13)

wherein each of Xn represents any single amino acid, though morepreferably represents an amino acid residue in the corresponding humanHDx sequences of the appended sequence listing.

A second motif, herein designated the χ motif is represented by theconsensus sequence:

CVXXXKXFXXPXXXXGGGGYTXRNVARXWXXET  (SEQ ID No. 14)

more preferably by the consensus sequence:

CVEX₈VKX₉FNX₁₀PLLX₁₁LGGGGYTX₁₂RNVARCWTYET  (SEQ ID No: 15)

wherein each of X_(n) represents any single amino acid, though morepreferably represents an amino acid residue in the corresponding humanHDx sequences of the appended sequence listing. The χ motif can be foundin the human HD1 sequence at C284-Thr316.

The family of HDx proteins apparently ranges in size from about 40 kd toabout 60 kd for the unmodified polypeptide chain. For instance, thebovine HD1 protein migrates on an SDS-PAGE (9%) gel with an apparentmolecular weight of 46 kD. The human HD1 amino acid sequence predicts amolecular weight for the polypeptide chain of 55 kD.

Accordingly, certain aspects of the present invention relate to nucleicacids encoding HDx proteins, the HDx proteins themselves, antibodiesimmunoreactive with HDx proteins, and preparations of such compositions.Moreover, the present invention provides diagnostic and therapeuticassays and reagents for detecting and treating disorders involving, forexample, aberrant expression (or loss thereof) of HDx homologs. Inaddition, drug discovery assays are provided for identifying agentswhich can modulate the biological function of HDx proteins, such as byaltering the binding of HDx molecules to either proteins or nucleicacids. Such agents can be useful therapeutically to alter the growthand/or differentiation of a cell. Other aspects of the invention aredescribed below or will be apparent to those skilled in the art in lightof the present disclosure.

Analysis of the human HDx sequences, while not revealing any obvioussimilarities to known domains or motifs, did indicate similarities withpreviously identified proteins from both Saccharomyces cerevisiae andXenopus laevis. Those genes, RPD3 (SEQ ID No. 9) and Xe-RPD3 (SEQ ID No.10), respectively, had not previously been ascribed any specificfunction. However, based on our observations for the function of HD1, itis now apparent that each of these other proteins are also deacetylases,and represent potential therapeutic targets. Accordingly, drug discoveryassays are provided for identifying agents which can modulate thebiological function of “HDx-related” proteins, such as RPD3 homologs, byaltering the enzymatic activity of the deacetylase, or its binding toother cellular components including homologs of RbAp48 (describedinfra). Such agents can be useful therapeutically to alter the growthand/or differentiation of non-human cells, such as in the treatment ofmycotic infections, or as additives to livestock feed, e.g., to promoteweight gain, or as topical antiseptics for sterilizing medicalequipment.

In addition we isolated another bovine protein having an approximatemolecular size of 50 kD which apparently binds HDx proteins isolated bythe trapoW n matrix, and microsequencing of that protein demonstratedthat it was related to the protein referred to in the art as RbAp48(Qian et al. (1993) Nature 364:648; SEQ ID NOS 11 & 17). RbAp48 wasoriginally identified as a protein that binds to the retinoblastoma (Rb)gene product. The retinoblastoma (RB) gene product plays a role in tumorsuppression (Weinberg, R. A., (September 1988) Scientific Amer. pp44-51; Hansen et al. (1988) Trends Genet 4:125-128). The role of RB as atumor-suppressor protein in cell-cycle control is believed to be similarto that of another tumor-suppressor, p53 (Green (1989) Cell 56:1-3;Mowat et al (1985 Nature 314:633-636). Inactivation or mutation of thesecond RB allele in one of the somatic cells of these susceptibleindividuals appears to be the molecular event that leads to tumorformation (Cavenee et al. (1983) Nature 305:799-784; Friend et al.(1987) PNAS 84:9059-9063).

The growth suppression function of the retinoblastoma protein is thoughtto be mediated by Rb binding to cellular proteins. RbAp48 is one of themajor proteins that binds to a putative functional domain at the carboxyterminus of the Rb protein. Complex formation between RbAp48 and Rboccurs in vitro and in vivo, and apparently involves direct interactionbetween the proteins. Like Rb, RbAp48 is a ubiquitously expressednuclear protein. RbAp48 share sequence homology with MSI1, a negativeregulator of the Ras-cyclic AMP pathway in the yeast Saccharomycescerevisiae. Furthermore, like MSIl, human RbAp48 suppresses theheat-shock sensitivity of the yeast iral strains and RAS2Val19 strains.Interaction with RbAp48 may be one of the mechanisms for suppression ofgrowth mediated by Rb. Accordingly, the interaction of RbAp48 with HDxproteins further implicates the HDx proteins in cell-cycle regulation.

The RpAp48 interaction with HDx and HDx-related proteins represents yetanother therapeutic target. Accordingly, drug discovery assays areprovided for identifying agents which can modulate the interaction ofRbAp48 proteins and the like with HDx-related proteins. Such assays canbe derived to detect the ability of a test agent to alterprotein-protein contacts, or to alter the enzymatic activity of thedeacetylase in complexes including an RbAp48 protein (e.g., were suchcomplexes allosterically modulate the HDx enzymatic activity). As above,such agents can be useful therapeutically to alter the growth and/ordifferentiation of cells.

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding one of the HDxpolypeptides of the present invention, including both exon and(optionally) intron sequences. A “recombinant gene” refers to nucleicacid encoding an HDx polypeptide and comprising HDx-encoding exonsequences, though it may optionally include intron sequences which areeither derived from a chromosomal HDx gene or from an unrelatedchromosomal gene. Exemplary recombinant genes encoding the subject HDxpolypeptide are represented in the appended Sequence Listing. The term“intron” refers to a DNA sequence present in a given HDx gene which isnot translated into protein and is generally found between exons.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of an HDx polypeptide or,where anti-sense expression occurs from the transferred gene, theexpression of a naturally-occurring form of the HDx protein isdisrupted.

As used herein, the term “specifically hybridizes” refers to the abilityof the probe/primer of the invention to hybridize to at least 15consecutive nucleotides of an HDx gene, such as an HDx sequencedesignated in one of SEQ ID Nos: 1-4, or a sequence complementarythereto, or naturally occurring mutants thereof, such that it has lessthan 15%, preferably less than 10%, and more preferably less than 5%background hybridization to a cellular nucleic acid (e.g., mRNA orgenomic DNA) encoding a protein other than an HDx protein, as definedherein. In preferred embodiments, the oligonucleotide probe specificallydetects only one of the subject HDx paralogs, e.g., does notsubstantially hybridize to transcripts for other HDx homologs in thesame species.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of one of the recombinant HDx genesis under the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of HDx genes.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue, such ascells of hepatic, pancreatic, neuronal or hematopoietic origin. The termalso covers so-called “leaky” promoters, which regulate expression of aselected DNA primarily in one tissue, but can cause at least low levelexpression in other tissues as well.

As used herein, a “transgenic animal” is any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the HDx proteins, e.g. either agonistic or antagonisticforms. However, transgenic animals in which the recombinant HDx gene issilent are also contemplated, as for example, the FLP or CRE recombinasedependent constructs described below. Moreover, “transgenic animal” alsoincludes those recombinant animals in which gene disruption of one ormore HDx genes is caused by human intervention, including bothrecombination and antisense techniques.

The “non-human animals” of the invention include vertebrates such asrodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The invention also contemplates transgenic insects,including those of the genus Drosophila, such as D. melanogaster. Theterm “chimeric animal” is used herein to refer to animals in which therecombinant gene is found, or in which the recombinant is expressed insome but not all cells of the animal. The term “tissue-specific chimericanimal” indicates that one of the recombinant HDx genes is presentand/or expressed or disrupted in some tissues but not others.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one of the HDx polypeptides, or pending an antisensetranscript thereto), which is partly or entirely heterologous, i.e.,foreign, to the transgenic animal or cell into which it is introduced,or, is homologous to an endogenous gene of the transgenic animal or cellinto which it is introduced, but which is designed to be inserted, or isinserted, into the animal's genome in such a way as to alter the genomeof the cell into which it is inserted (e.g., it is inserted at alocation which differs from that of the natural gene or its insertionresults in a knockout). A transgene can include one or moretranscriptional regulatory sequences and any other nucleic acid, such asintrons, that may be necessary for optimal expression of a selectednucleic acid.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term “DNAsequence encoding an HDx polypeptide” may thus refer to one or moregenes within a particular individual. Moreover, certain differences innucleotide sequences may exist between individuals of the same species,which are called alleles. Such allelic differences may or may not resultin differences in amino acid sequence of the encoded polypeptide yetstill encode a protein with the same biological activity.

“Homology” refers to sequence similarity between two peptides or betweentwo nucleic acid molecules. Homology can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences. An“unrelated” or “non-homologous” sequence shares less than 40 percentidentity, though preferably less than 25 percent identity, with one ofthe HDx sequences of the present invention.

As used herein, an “HDx-related” protein refers to the HDx proteinsdescribed herein, and other human homologs of those HDx sequences, aswell as orthologs and paralogs (homologs) of the HDx proteins in otherspecies, ranging from yeast to other mammals, e.g., homologous histonedeacetylase, The term “ortholog” refers to genes or proteins which arehomologs via speciation, e.g., closely related and assumed to havecommon descent based, on structural and functional considerations.Orthologous proteins function as recognizably the same activity indifferent species. The term “paralog” refers to genes or proteins whichare homologs via gene duplication, e.g., duplicated variants of a genewithin a genome. See also, Fritch, WM (1970) Syst Zool 19:99-113.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding one of the subject HDx polypeptides with a secondamino acid sequence defining a domain (e.g. polypeptide portion) foreignto and not substantially homologous with any domain of one of the HDxproteins. A chimeric protein may present a foreign domain which is found(albeit in a different protein) in an organism which also expresses thefirst protein, or it may be an “interspecies”, “intergenic”,etc. fusionof protein structures expressed by different kinds of organisms. Ingeneral, a fusion protein can be represented by the general formulaX-HDx-Y, wherein HDx represents a portion of the protein which isderived from one of the HDx proteins, and X and Y are, independently,absent or represent amino acid sequences which are not related to one ofthe HDx sequences in an organism.

The term “isolated” as also used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject HDx polypeptides preferably includes no more than 10 kilobases(kb) of nucleic acid sequence which naturally immediately flanks the HDxgene in genomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state.

As described below, one aspect of the invention pertains to isolatednucleic acids comprising nucleotide sequences encoding HDx polypeptides,and/or equivalents of such nucleic acids. The term nucleic acid as usedherein is intended to include fragments as equivalents. The termequivalent is understood to include nucleotide sequences encodingfunctionally equivalent HDx polypeptides or functionally equivalentpeptides having an activity of an HDx protein such as described herein.Equivalent nucleotide sequences will include sequences that differ byone or more nucleotide substitutions, additions or deletions, such asallelic variants; and will, therefore, include sequences that differfrom the nucleotide sequence of the HDx cDNA sequences shown in any ofSEQ ID Nos:1-4 due to the degeneracy of the genetic code. Equivalentswill also include nucleotide sequences that hybridize under stringentconditions (i.e., equivalent to about 20-27° C. below the meltingtemperature (T_(m)) of the DNA duplex formed in about 1M salt) to thenucleotide sequences represented in one or more of SEQ ID Nos:1-4. Inone embodiment, equivalents will further include nucleic acid sequencesderived from and evolutionarily related to, a nucleotide sequences shownin any of SEQ ID Nos: 1-4.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of one of thesubject HDx polypeptides which function in a limited capacity as one ofeither an HDx agonist (mimetic) or an HDx antagonist, in order topromote or inhibit only a subset of the biological activities of thenaturally-occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed to all of the biological activities ofnaturally occurring forms of HDx proteins.

Homologs of each of the subject HDx proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the HDx polypeptide from which it was derived. Alternatively,antagonistic forms of the protein can be generated which are able toinhibit the function of the naturally occurring form of the protein,such as by competitively binding to an HDx substrate or HDx associatedprotein, as for example competing with wild-type HDx in the binding ofRbAp48 or a histone. In addition, agonistic forms of the protein may begenerated which are constitutively active, or have an altered K_(cat) orK_(m) for deacetylation reactions. Thus, the HDx protein and homologsthereof provided by the subject invention may be either positive ornegative regulators of transcription and/or replication.

In general, polypeptides referred to herein as having an activity of anHDx protein (e.g., are “bioactive”) are defined as polypeptides whichinclude an amino acid sequence corresponding (e.g., identical orhomologous) to all or a portion of the amino acid sequences of an HDxproteins shown in any one or more of SEQ ID Nos:5-8 and which mimic orantagonize all or a portion of the biological/biochemical activities ofa naturally occurring HDx protein. Examples of such biological activityinclude the ability to modulate proliferation of cells. For example,inhibiting histone deacetylation causes cells to arrest in G1 and G2phases of the cell cycle. The biochemical activity associated with HDxproteins of the present invention can also characterized in terms ofbinding to and (optionally). catalyzing the deacetylation of anacetylated histone. Another biochemical property of certain of thesubject HDx proteins involves binding to other cellular proteins, suchas RbAp48.

Other biological activities of the subject HDx proteins are describedherein or will be reasonably apparent to those skilled in the art.According to the present invention, a polypeptide has biologicalactivity if it is a specific agonist or antagonist of anaturally-occurring form of an HDx protein.

Preferred nucleic acids encode an HDx polypeptide comprising an aminoacid sequence at least 80% homologous, more preferably at least 85%homologous and most preferably at least 88% homologous with an aminoacid sequence of a human HDx, e.g., such as selected from the groupconsisting of SEQ ID Nos: 5-8. Nucleic acids which encode polypeptidesat least about 90%, more preferably at least about 95%, and mostpreferably at least about 98-99% homology with an amino acid sequencerepresented in one of SEQ ID Nos:5-8 are of course also within the scopeof the invention, as are nucleic acids identical in sequence with any ofthe enumerated HDx sequences of the sequence listing. In one embodiment,the nucleic acid is a cDNA encoding a polypeptide having at least oneactivity of the subject HDx polypeptide.

In certain preferred embodiments, the invention features a purified orrecombinant HDx polypeptide having peptide chain with a molecular weightin the range of 40 kd to 60 kd, even more preferably in the range of45-50 kd or 53-58 kd. It will be understood that certainpost-translational modifications, e.g., phosphorylation and the like,can increase the apparent molecular weight of the HDx protein relativeto the unmodified polypeptide chain, and cleavage of certain sequences,such as pro-sequences, can likewise decrease the apparent molecularweight.

In other preferred embodiments, the nucleic acid encodes an HDxpolypeptide which includes both the ν and χ motifs, and preferablypossess a histone deacetylase activity. For example, preferred HDxproteins are represented by the general formula A-(νmotif)-B-(χmotif)-C, wherein the v motif is an amino acid sequence represented inSEQ ID No. 12, more preferably SEQ ID No. 13, the χ motif is an aminoacid sequence represented in SEQ ID No. 14, more preferably SEQ ID No.15, and A, B and C represent amino acid sequences which are correspondto HDx or HDx-related proteins.

Still other preferred nucleic acids of the present invention encode anHDx polypeptide which includes a polypeptide sequence corresponding toall or a portion of amino acid residues of any one of SEQ ID Nos: 5-8,e.g., at least 5, 10, 25, 50 or 100 amino acid residues of that region.

Another aspect of the invention provides a nucleic acid which hybridizesunder high or low stringency conditions to the nucleic acid representedby SEQ ID No: 1. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C.

Nucleic acids, having a sequence that differs from the nucleotidesequences shown in one of SEQ ID Nos: 1-4 due to degeneracy in thegenetic code are also within the scope of the invention. Such nucleicacids encode functionally equivalent peptides (i.e., a peptide having abiological activity of an HDx polypeptide) but differ in sequence fromthe sequence shown in the sequence listing due to degeneracy in thegenetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC each encode histidine) may result in“silent” mutations which do not affect the amino acid sequence of an HDxpolypeptide. However, it is expected that DNA sequence polymorphismsthat do lead to changes in the amino acid sequences of the subject HDxpolypeptides will exist among, for example, humans. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encodingpolypeptides having an activity of an HDx polypeptide may exist amongindividuals of a given species due to natural allelic variation.

As used herein, an HDx gene fragment refers to a nucleic acid havingfewer nucleotides than the nucleotide sequence encoding the entiremature form of an HDx protein yet which (preferably) encodes apolypeptide which retains some biological activity of the full lengthprotein. Fragment sizes contemplated by the present invention include,for example, 5, 10, 25, 50, 75, 100, or 200 amino acids in length.

As indicated by the examples set out below, HDx protein-encoding nucleicacids can be obtained from mRNA present in any of a number of eukaryoticcells. It should also be possible to obtain nucleic acids encoding HDxpolypeptides of the present invention from genomic DNA from both adultsand embryos. For example, a gene encoding an HDx protein can be clonedfrom either a cDNA or a genomic library in accordance with protocolsdescribed herein, as well as those generally known to persons skilled inthe art. A cDNA encoding an HDx protein can be obtained by isolatingtotal mRNA from a cell, e.g. a mammalian cell, e.g. a human cell,including embryonic cells. Double stranded cDNAs can then be preparedfrom the total mRNA, and subsequently inserted into a suitable plasmidor bacteriophage vector using any one of a number of known techniques.The gene encoding an HDx protein can also be cloned using establishedpolymerase chain reaction techniques in accordance with the nucleotidesequence information provided by the invention. The nucleic acid of theinvention can be DNA or RNA. A preferred nucleic acid is a cDNAincluding a nucleotide sequence represented by one of SEQ ID Nos: 1-4.

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotide probesor their derivatives which specifically hybridize (e.g. binds) undercellular conditions, with the cellular mRNA and/or genomic DNA encodingone or more of the subject HDx proteins so as to inhibit expression ofthat protein, e.g. by inhibiting transcription and/or translation. Thebinding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix. In general,“antisense” therapy refers to the range of techniques generally employedin the art, and includes any therapy which relies on specific binding tooligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes an HDx protein. Alternatively, the antisenseconstruct is an oligonucleotide probe which is generated ex vivo andwhich, when introduced into the cell causes inhibition of expression byhybridizing with the mRNA and/or genomic sequences of an HDx gene. Sucholigonucleotide probes are preferably modified oligonucleotides whichare resistant to endogenous nucleases, e.g. exonucleases and/orendonucleases, and are therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775), or peptide nucleic acids(PNAs). Additionally, general approaches to constructing oligomersuseful in antisense therapy have been reviewed, for example, by Van derKrol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988)Cancer Res 48:2659-2668.

Accordingly, the modified oligomers of the invention are useful intherapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of routes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remmington's PharmaceuticalSciences, Meade Publishing Co.; Easton, Pa. For systemic-administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous. For injection, the oligomers of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligomers may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of one of the HDx proteins,can be used in the manipulation of tissue, e.g. tissue differentiationor growth, both in vivo and ex vivo.

Furthermore, the anti-sense techniques (e.g. microinjection of antisensemolecules, or transfection with plasmids whose transcripts areanti-sense with regard to an HDx mRNA or gene sequence) can be used toinvestigate role of HDx in developmental events, as well as the normalcellular function of HDx in adult tissue. Such techniques can beutilized in cell culture, but can also be used in the creation oftransgenic animals (described infra).

This invention also provides expression vectors containing a nucleicacid encoding an HDx polypeptide, operably linked to at least onetranscriptional regulatory sequence. Operably linked is intended to meanthat the nucleotide sequence is linked to a regulatory sequence in amanner which allows expression of the nucleotide sequence. Regulatorysequences are art-recognized and are selected to direct expression ofthe subject HDx proteins. Accordingly, the term transcriptionalregulatory sequence includes promoters, enhancers and other expressioncontrol elements. Such regulatory sequences are described in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). For instance, any of a wide variety ofexpression control sequences, sequences that control the expression of aDNA sequence when operatively linked to it, may be used in these vectorsto express DNA sequences encoding HDx polypeptides of this invention.Such useful expression control sequences, include, for example, a viralLTR, such as the LTR of the Moloney murine leukemia virus, the early andlate promoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the. majoroperator and promoter regions of phage λ, the control regions for fdcoat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid. phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other proteins encoded by thevector, such as antibiotic markers, should also be considered. In oneembodiment, the expression vector includes a recombinant gene encoding apeptide having an agonistic activity of a subject HDx polypeptide, oralternatively, encoding a peptide which is an antagonistic form of theHDx protein, such as a catalytically-inactive deacetylase. Suchexpression vectors can be used to transfect cells and thereby producepolypeptides, including fusion proteins, encoded by nucleic acids asdescribed herein.

Moreover, the gene constructs of the present invention can also be usedas a part of a gene therapy protocol to deliver nucleic acids, e.g.,encoding either an agonistic or antagonistic form of one of the subjectHDx proteins or an antisense molecule described above. Thus, anotheraspect of the invention features expression vectors for in vivo or invitro transfection and expression of an HDx polypeptide or antisensemolecule in particular cell types so as to reconstitute the function of,or alternatively, abrogate the function of HDx-induced transcription ina tissue in which the naturally-occurring form of the protein ismisexpressed; or to deliver a form of the protein which altersdifferentiation of tissue, or which inhibits neoplastic transformation.

Expression constructs of the subject HDx polypeptides, as well asantisense constructs, may be administered in any biologically effectivecarrier, e.g. any formulation or composition capable of effectivelydelivering the recombinant gene to cells in vivo. Approaches includeinsertion of the subject gene in viral vectors including recombinantretroviruses, adenovirus, adeno-associated virus, and herpes simplexvirus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectorstransfect cells directly; plasmid DNA can be delivered with the help of,for example, cationic liposomes (lipofectin) or derivatized (e.g.antibody conjugated), polylysine conjugates, gramacidin S, artificialviral envelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. It will be appreciated that because transduction of appropriatetarget cells represents the critical first step in gene therapy, choiceof the particular gene delivery system will depend on such factors asthe phenotype of the intended target and the route of administration,e.g. locally or systemically. Furthermore, it will be recognized thatthe particular gene construct provided for in vivo transduction of HDxexpression are also useful for in vitro transduction of cells, such asfor use in the ex vivo tissue culture systems described below.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNAencoding the particular HDx polypeptide desired. Infection of cells witha viral vector has the advantage that a large proportion of the targetedcells can receive the nucleic acid. Additionally, molecules encodedwithin the viral vector, e.g., by a cDNA contained in the viral vector,are expressed efficiently in cells which have taken up viral vectornucleic acid. Retrovirus vectors, adenovirus vectors andadeno-associated virus vectors are exemplary recombinant gene deliverysystem for the transfer of exogenous genes in vivo, particularly intohumans. These vectors provide efficient delivery of genes into cells,and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a subjectHDx polypeptide in the tissue of an animal. Most nonviral methods ofgene transfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject HDx polypeptidegene by the targeted cell. Exemplary gene delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In clinical settings, the gene delivery systems for the therapeutic HDxgene can be introduced into a patient by any of a number of methods,each of which is familiar in the art. For instance, a pharmaceuticalpreparation of the gene delivery system can be introduced systemically,e.g. by intravenous injection, and specific transduction of the proteinin the target cells occurs predominantly from specificity oftransfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057). AN HDx gene, such as any one of the clonesrepresented in the group consisting of SEQ ID NO:1-4, can be deliveredin a gene therapy construct by electroporation using techniquesdescribed, for example, by Dev et al. ((1994) Cancer Treat Rev20:105-115).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery. system.

Another aspect of the present invention concerns recombinant forms ofthe HDx proteins. Recombinant polypeptides preferred by the presentinvention, in addition to native HDx proteins, are at least 80%homologous, more preferably at least 85% homologous and most preferablyat least 88% homologous with an amino acid sequence represented by anyof SEQ ID Nos: 5-8. Polypeptides which possess an activity of an HDxprotein (i.e. either agonistic or antagonistic), and which are at least90%, more preferably at least 95%, and most preferably at least about98-99% homologous with a sequence selected from the group consisting ofSEQ ID Nos: 5-8 are also within the scope of the invention. In otherpreferred embodiments, the HDx polypeptide includes both the ν and χmotifs, and preferably possess a histone deacetylase activity.

The term “recombinant HDx protein” refers to a polypeptide which isproduced by recombinant DNA techniques, wherein generally, DNA encodingan HDx polypeptide is inserted into a suitable expression vector whichis in turn used to transform a host cell to produce the heterologousprotein. Moreover, the phrase “derived from”, with respect to arecombinant HDx gene, is meant to include within the meaning of“recombinant protein”those proteins having an amino acid sequence of anative HDx protein, or an amino acid sequence similar thereto which isgenerated by mutations including substitutions and deletions (includingtruncation) of a naturally occurring form of the protein.

The present invention further pertains to recombinant forms of thesubject HDx polypeptides which are encoded by genes derived from amammal (e.g. a human), and which have amino acid sequencesevolutionarily related to the HDx proteins represented in SEQ ID Nos:5-8. Such recombinant HDx polypeptides preferably are capable offunctioning in one of either role of an agonist or antagonist of atleast one biological activity of a wild-type (“authentic”) HDx proteinof the appended sequence listing. The term “evolutionarily related to”,with respect to amino acid sequences of HDx proteins, refers to bothpolypeptides having amino acid sequences which have arisen naturally,and also to mutational variants of HDx polypeptides which are derived,for example, by combinatorial mutagenesis.

The present invention also provides methods of producing the subject HDxpolypeptides. For example, a host cell transfected with a nucleic acidvector directing expression of a nucleotide sequence encoding thesubject polypeptides can be cultured under appropriate conditions toallow expression of the peptide to occur. The cells may. be harvested,lysed and the protein isolated. A cell culture includes host cells,media and other byproducts. Suitable media for cell culture are wellknown in the art. The recombinant HDx polypeptide can be isolated fromcell culture medium, host cells, or both using techniques known in theart for purifying proteins including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for such peptide.In a preferred embodiment, the recombinant HDx polypeptide is a fusionprotein containing a domain which facilitates its purification, such asGST fusion protein or poly(His) fusion protein.

This invention also pertains to a host cell transfected to expressrecombinant forms of the subject HDx polypeptides. The host cell may beany prokaryotic or eukaryotic cell. Thus, a nucleotide sequence derivedfrom the cloning of HDx proteins, encoding all or a selected portion ofa full-length protein, can be used to produce a recombinant form of anHDx polypeptide via microbial or eukaryotic cellular processes. Ligatingthe polynucleotide sequence into a gene construct, such as an expressionvector, and transforming or transfecting into hosts, either eukaryotic(yeast, avian, insect or mammalian) or prokaryotic (bacterial cells),are standard procedures used in producing other well-known proteins,e.g. MAP kinases, p53, WT1, PTP phosphatases, SRC, and the like. Similarprocedures, or modifications thereof, can be employed to preparerecombinant HDx polypeptides by microbial means or tissue-culturetechnology in accord with the subject invention.

The recombinant HDx genes can be produced by ligating nucleic acidencoding an HDx protein, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells, or both.Expression vectors for production of recombinant forms of the subjectHDx polypeptides include plasmids and other vectors. For instance,suitable vectors for the expression of an HDx polypeptide includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused. In an illustrative embodiment, an HDx polypeptide is producedrecombinantly utilizing an expression vector generated by sub-cloningthe coding sequence of one of the HDx genes represented in SEQ IDNos:1-4.

The preferred mammalian expression vectors contain both prokaryoticsequences, to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRC/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papillomavirus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Thevarious methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

In some instances, it may be desirable to express the recombinant HDxpolypeptide by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

When it is desirable to express only a portion of an HDx protein, suchas a form lacking a portion of the N-terminus, i.e. a truncation mutantwhich lacks the signal peptide, it may be necessary to add a start codon(ATG) to the oligonucleotide fragment containing the desired sequence tobe expressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing HDx-derived polypeptides in a hostwhich produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitroby use of purified MAP (e.g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable to produce an immunogenicfragment of an HDx protein. For example, the VP6 capsid protein ofrotavirus can be used as an immunologic carrier protein for portions ofthe HDx polypeptide, either in the monomeric form or in the form of aviral particle. The nucleic acid sequences corresponding to the portionof a subject HDx protein to which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising HDxepitopes as part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the Hepatitis B surface antigenfusion proteins that recombinant Hepatitis B virions can be utilized inthis role as well. Similarly, chimeric constructs coding for fusionproteins containing a portion of an HDx protein and the polioviruscapsid protein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No: 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canalso be utilized to generate an immunogen, wherein a desired portion ofan HDx polypeptide is obtained directly from organo-chemical synthesisof the peptide onto an oligomeric branching lysine core (see, forexample, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992)J. Immunol. 148:914). Antigenic determinants of HDx proteins can also beexpressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, and accordingly, can be used in the expressionof the HDx polypeptides of the present invention. For example, HDxpolypeptides can be generated as glutathione-S-transferase (GST-fusion)proteins. Such GST-fusion proteins can enable easy purification of theHDx polypeptide, as for example by the use of glutathione-derivatizedmatrices (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni2+ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified protein (e.g., see Hochuli et al. (1987) J.Chromatography 411:177; and Janknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

HDx polypeptides may also be chemically modified to create HDxderivatives by forming covalent or aggregate conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of HDx proteins can beprepared by linking the chemical moieties to functional groups on aminoacid sidechains of the protein or at the N-terminus or at the C-terminusof the polypeptide.

The present invention also makes available isolated HDx polypeptideswhich are isolated from, or otherwise substantially free of othercellular proteins, especially other signal transduction factors and/ortranscription factors which may normally be associated with the HDxpolypeptide. The term “substantially free of other cellular proteins”(also referred to herein as “contaminating proteins”) or “substantiallypure or purified preparations” are defined as encompassing preparationsof HDx polypeptides having less than 20% (by dry weight) contaminatingprotein, and preferably having less than 5% contaminating protein.Functional forms of the subject polypeptides can be prepared, for thefirst time, as purified preparations by using a cloned gene as describedherein. By “purified”, it is meant, when referring to a peptide or DNAor RNA sequence, that the indicated molecule is present in thesubstantial absence of other biological macromolecules, such as otherproteins. The term “purified” as used herein preferably means at least80% by dry weight, more preferably in the range of 95-99% by weight, andmost preferably at least 99.8% by weight, of biological macromoleculesof the same type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “opure” as used herein preferably has the samenumerical limits as “purified” immediately above. “Isolated” and“purified” do not encompass either natural materials in their nativestate or natural materials that have been separated into components(e.g., in an acrylamide gel) but not obtained either as pure (e.g.lacking contaminating proteins, or chromatography reagents such asdenaturing agents and polymers, e.g. acrylamide or agarose) substancesor solutions. In preferred embodiments, purified HDx preparations willlack any contaminating proteins from the same animal from that HDx isnormally produced, as can be accomplished by recombinant expression of,for example, a human HDx protein in a non-human cell.

As described above for recombinant polypeptides, isolated HDxpolypeptides can include all or a portion of an amino acid sequencescorresponding to an HDx polypeptide represented in any one of SEQ IDNos: 5-8 or homologous sequences thereto. In preferred embodiments, theHDx polypeptide includes both the ν and χ motifs, and preferably possessa histone deacetylase activity.

Isolated peptidyl portions of HDx proteins can be obtained by screeningpeptides recombinantly produced from the corresponding fragment of thenucleic acid encoding such peptides. In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Forexample, an HDx polypeptide of the present invention may be arbitrarilydivided into fragments of desired length with no overlap of thefragments, or preferably divided into overlapping fragments of a desiredlength. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments which canfunction as either agonists or antagonists of a wild-type (e.g.,“authentic”) HDx protein.

The recombinant HDx polypeptides of the present invention also includehomologs of the authentic HDx proteins, such as versions of thoseprotein which are resistant to proteolytic cleavage, as for example, dueto mutations which alter ubiquitination or other enzymatic targetingassociated with the protein.

Modification of the structure of the subject HDx polypeptides can be forsuch purposes as enhancing therapeutic or prophylactic efficacy,stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo), or post-translational modifications (e.g., toalter phosphorylation pattern of protein). Such modified peptides, whendesigned to retain at least one activity of the naturally-occurring formof the protein, or to produce specific antagonists thereof, areconsidered functional equivalents of the HDx polypeptides described inmore detail herein. Such modified peptides can be produced, forinstance, by amino acid substitution, deletion, or addition.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. isosteric and/or isoelectricmutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino acids that are related in their sidechains. Genetically encoded amino acids are can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutanine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. In similar fashion, the amino acid repertoirecan be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine,isoleucine, serine, threonine, with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methionine. (see, for example,Biochemistry, 2nd ed., Ed. by L. Stryer, WH Freeman and Co.: 1981).Whether a change in the amino acid sequence of a peptide results in afunctional HDx homolog (e.g. functional in the sense that the resultingpolypeptide mimics or antagonizes the wild-type form) can be readilydetermined by assessing the ability of the variant peptide to produce aresponse in cells in a fashion similar to the wild-type protein, orcompetitively inhibit such a response. Polypeptides in which more thanone replacement has taken place can readily be tested in the samemanner.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the subject HDx proteins as well as truncationmutants, and is especially useful for identifying potential variantsequences (e.g. homologs) that are functional in modulating histonedeacetylation. The purpose of screening such combinatorial libraries isto generate, for example, novel HDx homologs which can act as eitheragonists or antagonist, or alternatively, possess novel activities alltogether. To illustrate, HDx homologs can be engineered by the presentmethod to provide selective, constitutive activation of enzymaticactivity. Thus, combinatorially-derived homologs can be generated tohave an increased potency relative to a naturally occurring form of theprotein.

Likewise, HDx homologs can be generated by the present combinatorialapproach to selectively inhibit (antagonize) histone deacetylation. Forinstance, mutagenesis can provide HDx homologs which are able to bindother regulatory proteins or cytoskeletal elements (or DNA) yet preventacetylation of histones, e.g. the homologs can be dominant negativemutants. In a preferred embodiment, a dominant negative mutant of an HDxprotein is mutated at one or more residues of its catalytic site and/orspecificity subsites.

In one aspect of this method, the amino acid sequences for a populationof HDx homologs or other related proteins are aligned, preferably topromote the highest homology possible. Such a population of variants caninclude, for example, HDx homologs from one or more species. Amino acidswhich appear at each position of the aligned sequences are selected tocreate a degenerate set of combinatorial sequences. In a preferredembodiment, the variegated library of HDx variants is generated bycombinatorial mutagenesis at the nucleic acid level, and is encoded by avariegated gene library. For instance, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential HDx sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g. for phage display) containing the set of HDx sequencestherein.

As illustrated in FIG. 5B, to analyze the sequences of a population ofvariants, the amino acid sequences of interest can be aligned relativeto sequence homology. The presence or absence of amino acids from analigned sequence of a particular variant is relative to a chosenconsensus length of a reference sequence, which can be real orartificial. For instance, FIG. 5B includes the alignment of the ν andχ-motifs for several of the HDx gene products. Analysis of the alignmentof these sequences from the clones can give rise to the generation of adegenerate library of polypeptides comprising potential HDx sequences.In an exemplary embodiment, a library of variants based on the HD1sequence, but degenerate across each of the ν and χ-motifs can beprovided. On such library can be represented by the general formula A-(νmotif)-B-(χ motif)-C, wherein the ν motif is an amino acid sequencerepresented in the general formula

DIAX1NWAGGLHHAKKX2EASGFCYVNDIVX3X4ILELLKYHX5RVLYIDIDIHHGDGX6EAFYX7TD-RVMTVSF  (SEQ ID No. 13)

The χ motif is an amino acid sequence represented in the general formula

CVEX8VKX9FNX10PLLX11LGGGGYTX12RNVARCWTYET  (SEQ ID No. 15)

A corresponds to Met1-Thr129 of SEQ ID No. 5, B corresponds toHis199-Lys283 of SEQ ID No. 5, and C correponds to Ala317-Ala482 of SEQID No. 5, wherein X₁ represents Ile or Val; X₂ represents Phe or Ser; X₃represents Phe or Leu; X₄ represents Gly or Ala; X₅ represents Pro orGin; X₆ represents Gin or Glu; X₇ represents Leu or Thr; X₈ representsVal or Tyr; X₉ represents Thr or Ser; X₁₀ represents Leu or Ile; X₁₁,represents Met or Val; and X₁₂ represents Ile or Val. To further expandthe combinatorial set, other conservative mutations relative to thoseappearing in the human sequences can be provided. For example, in a moreexpansive library, X₁ represents Gly, Ala, Val, Ile or Leu; X₂represents Phe, Tyr, Thr or Ser; X₃ represents Phe, Tyr, Gly, Ala, Val,Ile or Leu; X₄ represents Gly, Ala, Val, Ile or Leu; X₅ represents Pro,Asn or Gin; X₆ represents Asn, Gin, Asp or Glu; X₇ represents Gly, Ala,Val, Ile, Leu, Ser or Thr; X₈ represents Gly, Ala, Val, Ile, Leu, Phe orTyr; X₉ represents Thr, Cys, or Ser; X₁₀ represents Gly, Ala, Val, Ileor Leu; Xl represents Met, Cys, Gly, Ala, Val, Ile, Leu, Ser or Thr; andX₁₂ represents Gly, Ala, Val, Ile or Leu. In still another library, eachdegenerate position can be any one of the naturally occurring aminoacids. Likewise, the ν and χ-motifs can correspond to the degeneratesequences designated by SEQ ID Nos. 12 and 14, respectively.

There are many ways by which such libraries of potential HDx homologscan be generated from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic genes then ligated into anappropriate expression vector. The purpose of a degenerate set of genesis to provide, in one mixture, all of the sequences encoding the desiredset of potential HDx sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rdCleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevierpp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura etal. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al. (1990) Science 249:386-390;Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S.Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Likewise, a library of coding sequence fragments can be provided for anHDx clone in order to generate a variegated population of HDx fragmentsfor screening and subsequent selection of bioactive fragments. A varietyof techniques are known in the art for generating such libraries,including chemical synthesis. In one embodiment, a library of codingsequence fragments can be generated by (i) treating a double strandedPCR fragment of an HDx coding sequence with a nuclease under conditionswherein nicking occurs only about once per molecule; (ii) denaturing thedouble stranded DNA; (iii) renaturing the DNA to form double strandedDNA which can include sense/antisense pairs from different nickedproducts; (iv) removing single stranded portions from reformed duplexesby treatment with S1 nuclease; and (v) ligating the resulting fragmentlibrary into an expression vector. By this exemplary method, anexpression library can be derived which codes for N-terminal, C-terminaland internal fragments of various sizes.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of HDx homologs. The most widely used techniques forscreening large gene libraries typically comprises cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected.

In an exemplary embodiment, the library of HDx variants is expressed asa fusion protein on the surface of a viral particle. For instance, inthe filamentous phage system, foreign peptide sequences can be expressedon the surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, a large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd., and fl are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461).

For example, the recombinant phage antibody system (RPAS, PharmaciaCatalog number 27-9400-01) can be easily modified for use in expressingand screening HDx combinatorial libraries by panning on glutathioneimmobilized histones/GST fusion proteins or RbAp48/GST fusion protein toenrich for HDx homologs which retain an ability to bind a substrate orregulatory protein. Each of these HDx homologs can subsequently bescreened for further biological activities in order to differentiateagonists and antagonists. For example, histone-binding homologs isolatedfrom the combinatorial library can be tested for their enzymaticactivity directly, or for their effect on cellular proliferationrelative to the wild-type form of the protein.

The invention also provides for reduction of the HDx or RbAp48 orhistones proteins to generate mimetics, e.g. peptide or non-peptideagents, which are able to disrupt a biological activity of an HDxpolypeptide of the present invention, e.g. as catalytic inhibitor or aninhibitor of protein-protein interactions. Thus, such mutagenictechniques as described above are also useful to map the determinants ofthe HDx proteins which participate in protein-protein or protein-DNAinteractions involved in, for example, interaction of the subject HDxpolypeptide with histones, RbAp48 or cytoskeletal elements. Toillustrate, the critical residues of a subject HDx polypeptide which areinvolved in molecular recognition of histones can be determined and usedto generate HDx-derived peptidomimetics which competitively inhibitbinding of the authentic HDx protein with that moiety. Likewise,residues of a histone or of RbAp48 involved in binding to HDx proteinscan be identified, and peptides or peptidomimetics based on suchresidues can also be used as competitive inhibitors of the interactionof an HDx protein with either of those proteins. By employing, forexample, scanning mutagenesis to map the amino acid residues of aprotein which is involved in binding other proteins, peptidomimeticcompounds can be generated which mimic those residues which facilitatethe interaction. Such mimetics may then be used to interfere with thenormal function of an HDx protein. For instance, non-hydrolyzablepeptide analogs of such residues can be generated using benzodiazepine(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine(e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295;and Ewenson et al. in Peptides: Structure and Function (Proceedings ofthe 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.,1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), andβ-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71).

Another aspect of the invention pertains to an antibody specificallyreactive with an HDx protein. For example, by using immunogens derivedfrom an HDx protein, e.g. based on the cDNA sequences,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols (See, for example, Antibodies: A Laboratory Manualed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, suchas a mouse, a hamster or rabbit can be immunized with an immunogenicform of the peptide (e.g., an HDx polypeptide or an antigenic fragmentwhich is capable of eliciting an antibody response). Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers or other techniques well known in the art. An immunogenicportion of an HDx protein can be administered in the presence ofadjuvant. The progress of immunization can be monitored by detection ofantibody titers in plasma or serum. Standard ELISA or other immunoassayscan be used with the immunogen as antigen to assess the levels ofantibodies. In a preferred embodiment, the subject antibodies areimmunospecific for antigenic determinants of an HDx protein of aorganism, such as a mammal, e.g. antigenic determinants of a proteinrepresented by one of SEQ ID Nos: 5-8 or closely related homologs (e.g.at least 85% homologous, preferably at least 90% homologous, and morepreferably at least 95% homologous). In yet a further preferredembodiment of the present invention, in order to provide, for example,antibodies which are immuno-selective for discrete HDx homologs, e.g.HD1, the anti-HDx polypeptide antibodies do not substantially crossreact (i.e. does not react specifically) with a protein which is, forexample, less than 85%, 90% or 95% homologous with the selected HDx. By“not substantially cross react”, it is meant that the antibody has abinding affinity for a non-homologous protein which is at least oneorder of magnitude, more preferably at least 2 orders of magnitude, andeven more preferably at least 3 orders of magnitude less than thebinding affinity of the antibody for the intended target HDx.

Following immunization of an animal with an antigenic preparation of anHDx polypeptide, anti-HDx antisera can be obtained and, if desired,polyclonal anti-HDx antibodies isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (I975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an HDx polypeptideof the present invention and monoclonal antibodies isolated from aculture comprising such hybridoma cells.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subject HDxpolypeptides. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present invention is further intended toinclude bispecific and chimeric molecules having affinity for an HDxprotein conferred by at least one CDR region of the antibody.

Both monoclonal and polyclonal antibodies (Ab) directed againstauthentic HDx polypeptides, or HDx variants, and antibody fragments suchas Fab, F(ab)₂, Fv and scFv can be used to block the action of one ormore HDx proteins and allow the study of the role of these proteins in,for example, differentiation of tissue. Experiments of this nature canaid in deciphering the role of HDx proteins that may be involved incontrol of proliferation versus differentiation, e.g., in patterning andtissue formation.

Antibodies which specifically bind HDx epitopes can also be used inimmunohistochemical staining of tissue samples in order to evaluate theabundance and pattern of expression of each of the subject HDxpolypeptides. Anti-HDx antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate HDxprotein levels in tissue as part of a clinical testing procedure. Forinstance, such measurements can be useful in predictive valuations ofthe onset or progression of proliferative or differentiative disorders.Likewise, the ability to monitor HDx protein levels in an individual canallow determination of the efficacy of a given treatment regimen for anindividual afflicted with such a disorder. The level of HDx polypeptidesmay be measured from cells in bodily fluid, such as in samples ofcerebral spinal fluid or amniotic fluid, or can be measured in tissue,such as produced by biopsy. Diagnostic assays using anti-HDx antibodiescan include, for example, immunoassays designed to aid in earlydiagnosis of a disorder, particularly ones which are manifest at birth.Diagnostic assays using anti-HDx polypeptide antibodies can also includeimmunoassays designed to aid in early diagnosis and phenotypingneoplastic or hyperplastic disorders.

Another application of anti-HDx antibodies of the present invention isin the immunological screening of cDNA libraries constructed inexpression vectors such as λgt11, λgt18-23, λZAP, and λORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, λgt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of an HDx protein,e.g. other orthologs of a particular HDx protein or other paralogs fromthe same species, can then be detected with antibodies, as, for example,reacting nitrocellulose filters lifted from infected plates withanti-HDx antibodies. Positive phage detected by this assay can then beisolated from the infected plate. Thus, the presence of HDx homologs canbe detected and cloned from other animals, as can alternate isoforms(including splicing variants) from humans.

Moreover, the nucleotide sequences determined from the cloning of HDxgenes from organisms will further allow for the generation of probes andprimers designed for use in identifying and/or cloning HDx homologs inother cell types, e.g. from other tissues, as well as HDx homologs fromother organisms. For instance, the present invention also provides aprobe/primer comprising a substantially purified oligonucleotide, whicholigonucleotide comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least 10 consecutivenucleotides of sense or anti-sense sequence selected from the groupconsisting of SEQ ID Nos: 1-4 or naturally occurring mutants thereof.For instance, primers based on the nucleic acid represented in SEQ IDNos: 1-4 can be used in PCR reactions to clone HDx homologs. Likewise,probes based on the subject HDx sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto and able to be detected, e.g. the label group isselected from amongst radioisotopes, fluorescent compounds, enzymes, andenzyme co-factors.

Such probes can also be used as a part of a diagnostic test kit foridentifying cells or tissue which misexpress an HDx protein, such as bymeasuring a level of an HDx-encoding nucleic acid in a sample of cellsfrom a patient; e.g. detecting HDx mRNA levels or determining whether agenomic HDx gene has been mutated or deleted.

To illustrate, nucleotide probes can be generated from the subject HDxgenes which facilitate histological screening of intact tissue andtissue samples for the presence (or absence) of HDx-encodingtranscripts. Similar to the diagnostic uses of anti-HDx antibodies, theuse of probes directed to HDx messages, or to genomic HDx sequences, canbe used for both predictive and therapeutic evaluation of allelicmutations which might be manifest in, for example, neoplastic orhyperplastic disorders (e.g. unwanted cell growth) or abnormaldifferentiation of tissue. Used in conjunction with immunoassays asdescribed above, the oligonucleotide probes can help facilitate thedetermination of the molecular basis for a developmental disorder whichmay involve some abnormality associated with expression (or lackthereof) of an HDx protein. For instance, variation in polypeptidesynthesis can be differentiated from a mutation in a coding sequence.

Accordingly, the present method provides a method for determining if asubject is at risk for a disorder characterized by aberrant cellproliferation and/or differentiation. In preferred embodiments, methodcan be generally characterized as comprising detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesioncharacterized by at least one of (i) an alteration affecting theintegrity of a gene encoding an HDx-protein, or (ii) the mis-expressionof the HDx gene. To illustrate, such genetic lesions can be detected byascertaining the existence of at least one of (i) a deletion of one ormore nucleotides from an HDx gene, (ii) an addition of one or morenucleotides to an HDx gene, (iii) a substitution of one or morenucleotides of an HDx gene, (iv) a gross chromosomal rearrangement of anHDx gene, (v) a gross alteration in the level of a messenger RNAtranscript of an HDx gene, (vii) aberrant modification of an HDx gene,such as of the methylation pattern of the genomic DNA, (vii) thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of an HDx gene, (viii) a non-wild type level of anHDx-protein, and (ix) inappropriate post-translational modification ofan HDx-protein. As set out below, the present invention provides a largenumber of assay techniques for detecting lesions in an HDx gene, andimportantly, provides the ability to discern between different molecularcauses underlying HDx-dependent aberrant cell growth, proliferationand/or differentiation.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a (purified) oligonucleotide probe including a region ofnucleotide sequence which is capable of hybridizing to a sense orantisense sequence of an HDx gene, such as represented by any of SEQ IDNos: 1-4, or naturally occurring mutants thereof, or 5′ or 3′ flankingsequences or intronic sequences naturally associated with the subjectHDx genes or naturally occurring mutants thereof. The nucleic acid of acell is rendered accessible for hybridization, the probe is exposed tonucleic acid of the sample, and the hybridization of the probe to thesample nucleic acid is detected. Such techniques can be used to detectlesions at either the genomic or mRNA level, including deletions,substitutions, etc., as well as to determine mRNA transcript levels.

In certain embodiments, detection of the lesion comprises utilizing theprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1944) PNAS91:360-364), the later of which can be particularly useful for detectingpoint mutations in the HDx gene. In a merely illustrative embodiment,the method includes the steps of (i) collecting a sample of cells from apatient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) fromthe cells of the sample, (iii) contacting the nucleic acid sample withone or more primers which specifically hybridize to an HDx gene underconditions such that hybridization and amplification of the HDx gene (ifpresent) occurs, and (iv) detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample.

In still another embodiment, the level of an HDx-protein can be detectedby: immunoassay. For instance, the cells of a biopsy sample can belysed, and the level of an HDx-protein present in the cell can bequantitated by standard immunoassay techniques. In yet another exemplaryembodiment, aberrant methylation patterns of an HDx gene can be detectedby digesting genomic DNA from a patient sample with one or morerestriction endonucleases that are sensitive to methylation and forwhich recognition sites exist in the HDx gene (including in the flankingand intronic sequences). See, for example, Buiting et al. (1994) HumanMol Genet 3:893-895. Digested DNA is separated by gel electrophoresis,and hybridized with probes derived from, for example, genomic or cDNAsequences. The methylation status of the HDx gene can be determined bycomparison of the restriction pattern generated from the sample DNA withthat for a standard of known methylation.

In yet another aspect of the invention, the subject HDx polypeptides canbe used to generate a “two hybrid” assay or an “interaction trap” assay(see, for example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel etal. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), for isolating coding sequences forother cellular proteins which bind HDxs (“HDx-binding proteins” or“HDx-bp”). Such HDx-binding proteins would likely be involved in theregulation of HDx, e.g., as regulatory subunits or transducers, or besubstrates which are regulated by an HDx.

Briefly, the interaction trap relies on reconstituting in vivo afunctional transcriptional activator protein from two separate fusionproteins. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. To illustrate, a first hybrid gene comprisesthe coding sequence for a DNA-binding domain of a transcriptionalactivator fused in frame to the coding sequence for an HDx polypeptide.The second hybrid protein encodes a transcriptional activation domainfused in frame to a sample gene from a cDNA library. If the bait andsample hybrid proteins are able to interact, e.g., form an HDx-dependentcomplex, they bring into close proximity the two domains of thetranscriptional activator. This proximity is sufficient to causetranscription of a reporter gene which is operably linked to atranscriptional regulatory site responsive to the transcriptionalactivator, and expression of the reporter gene can be detected and usedto score for the interaction of the HDx and sample proteins.

Furthermore, by making available purified and recombinant HDxpolypeptides, the present invention facilitates the development ofassays which can be used to screen for drugs, including HDx homologs,which are either agonists or antagonists of the normal cellular functionof the subject HDx polypeptides, or of their role in the pathogenesis ofcellular differentiation and/or proliferation and disorders relatedthereto. Moreover, because we have also identified HDx-related proteins,such as the yeast RPD3 proteins, as histone deacetylases, the presentinvention further provides drug screening assays for detecting agentswhich modulate the bioactivity of HDx-related proteins. Such agents,when directed. to, for example, fungal HDx-related proteins, can be usedin the treatment of various infections. In a general sense, the assayevaluates the ability of a compound to modulate binding between an HDxpolypeptide and a molecule, be it protein or DNA, that interacts withthe HDx polypeptide. It will be apparent from the following descriptionof exemplary assays that, in place of a human HDx protein, the assay canbe derived with an HDx-related protein such as RPD3. Likewise, in placeof human RbAp48, other HDx-binding proteins can be used, e.g., otherhuman proteins. Exemplary compounds which can be screened includepeptides, nucleic acids, carbohydrates, small organic molecules, andnatural product extract libraries, such as isolated from animals,plants, fungus and/or microbes.

It is contemplated that any of the novel interactions described hereincould be exploited in a drug screening assay. For example, in oneembodiment, the interaction between an HDx protein and RbAp48 can bedetected in the presence and the absence of a test compound. In anotherembodiment, the ability of a compound to modulate the binding of an HDxprotein, or HDx-related protein such as the yeast RPD3, with histonescan be assessed. The identification of a test compound which influences,for example, HD1 catalyzed deacetylation of histones would be useful inthe modulation of HD1 activity in mammalian cells, while theidentification of a test compound which selectively inhibits the yeastRPD3 deacetylase activity would be useful as an antifungal agent. Inother embodiments the effect of a test compound on the binding of an HDxprotein to other molecules, such as cytoskeletal components, or otherproteins identified by the HDx-dependent ITS set out above, could betested. A variety of assay formats will suffice and, in light of thepresent inventions, will be comprehended by a skilled artisan.

In a preferred embodiment, assays which employ the subject mammalian HDxproteins can be used to identify compounds that have therapeutic indexesmore favorable than sodium butyrate, trapoxin, trichostatin or the like.For instance, trapoxin-like drugs can be identified by the presentinvention which have enhanced tissue-type or cell-type specificityrelative to trapoxin. To illustrate, the subject assays can be used togenerate compounds which preferentially inhibit IL-2 mediatedproliferation/activation of lymphocytes, or inhibit proliferation ofcertain tumor cells, without substantially interfering with othertissues, e.g. hepatocytes. Likewise, similar assays can be used toidentify drugs which inhibit proliferation of yeast cells or other lowereukaryotes, but which have a substantially reduced effect on mammaliancells, thereby improving therapeutic index of the drug as ananti-mycotic agent.

In one embodiment, the identification of such compounds is made possibleby the use of differential screening assays which detect and comparedrug-mediated inhibition of deacetylase activity between two or moredifferent HDx-like enzymes, or compare drug-mediated inhibition offormation of complexes involving two or more different types of HDx-likeproteins. To illustrate, the assay can be designed for side-by-sidecomparison of the effect of a test compound on the deacetylase activityor protein interactions of tissue-type specific HDx proteins. Given theapparent diversity of HDx proteins, it is probable that differentfunctional HDx activities, or HDx complexes exist and, in certaininstances, are localized to particular tissue or cell types. Thus, testcompounds can be screened for agents able to inhibit the tissue-specificformation of only a subset of the possible repertoire of HDx/regulatoryprotein complexes, or which preferentially inhibit certain HDx enzymes.In an exemplary embodiment, an interaction trap assay can be derivedusing two or more different human HDx “bait” proteins, while the “fish”protein is constant in each, e.g. a human RbAp48 construct. Running theinteraction trap side-by-side permits the detection of agents which havea greater effect (e.g. statistically significant) on the formation ofone of the HDx/RbAp48 complexes than on the formation of the other HDxcomplexes.

In similar fashion, differential screening assays can be used to exploitthe difference in protein interactions and/or catalytic mechanism ofmammalian HDx proteins and yeast RPD3 proteins in order to identifyagents which display a statistically significant increase in specificityfor inhibiting the yeast enzyme relative to the mammalian enzyme. Thus,lead compounds which act specifically on pathogens, such as fungusinvolved in mycotic infections, can be developed. By way ofillustration, the present assays can be used to screen for agents whichmay ultimately be useful for inhibiting at least one fungus implicatedin such mycosis as candidiasis, aspergillosis, mucormycosis,blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis,coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis,rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis,monoliasis, or sporotrichosis. For example, if the mycotic infection towhich treatment is desired is candidiasis, the present assay cancomprise comparing the relative effectiveness of a test compound oninhibiting the deacetylase activity of a mammalian HDx protein with itseffectiveness towards inhibiting the deacetylase activity of an RPD3homolog cloned from yeast selected from the group consisting of Candidaalbicans, Candida stellatoidea, Candida tropicalis, Candidaparapsilosis, Candida krusei, Candida pseudotropicalis, Candidaquillermondii, or Candida rugosa. Likewise, the present assay can beused to identify anti-fungal agents which may have therapeutic value inthe treatment of aspergillosis by selectively targeting RPD3 homologscloned from yeast such as Aspergillus fumigatus, Aspergillus flavus,Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus. Wherethe mycotic infection is mucormycosis, the RPD3 deacetylase can bederived from yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidiacorymbifera, Absidia ramosa, or Mucor pusillus. Sources of other RPD3activities for comparison with a mammalian HDx activity includes thepathogen Pneumocystis carinii.

In addition to such HDx therapeutic uses, anti-fungal agents developedwith such differential screening assays can be used, for example, aspreservatives in foodstuff, feed supplement for promoting weight gain inlivestock, or in disinfectant formulations for treatment of non-livingmatter, e.g., for decontaminating hospital equipment and rooms.

In similar fashion, side by side comparison of inhibition of a mammalianHDx proteins and an insect HDx-related proteins, will permit selectionof HDx inhibitors which discriminate between the human/mammalian andinsect enzymes. Accordingly, the present invention expresslycontemplates the use and formulations of the subject HDx therapeutics ininsecticides, such as for use in management of insects like the fruitfly.

In yet another embodiment, certain of the subject HDx inhibitors can beselected on the basis of inhibitory specificity for plant HDx-relatedactivities relative to the mammalian enzyme. For example, a plantHDx-related protein can be disposed in a differential screen with one ormore of the human enzymes to select those compounds of greatestselectivity for inhibiting the plant enzyme. Thus, the present inventionspecifically contemplates formulations of the subject HDx inhibitors foragricultural applications, such as in the form of a defoliant or thelike.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements. Accordingly, in an exemplary screening assay of thepresent invention, a reaction mixture is generated to include an HDxpolypeptide, compound(s) of interest, and a “target polypeptide”, e.g.,a protein, which interacts with the HDx polypeptide, whether as asubstrate or by some other protein-protein interaction. Exemplary targetpolypeptides include histones and RbAp48 polypeptides. Detection andquantification of complexes containing the HDx protein provide a meansfor determining a compound's efficacy at inhibiting (or potentiating)complex formation between the HDx and the target polypeptide. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. In the control assay, isolated and purified HDxpolypeptide is added to a composition containing the target polypeptideand the formation of a complex is quantitated in the absence of the testcompound.

Complex formation between the HDx polypeptide and the target polypeptidemay be detected by a variety of techniques. Modulation of the formationof complexes can be quantitated using, for example, detectably labeledproteins such as radiolabeled, fluorescently labeled, or enzymaticallylabeled HDx polypeptides, by immunoassay, by chromatographic detection,or by detecting the intrinsic activity of the acetylase.

Typically, it will be desirable to immobilize either HDx or the targetpolypeptide to facilitate separation of complexes from uncomplexed formsof one or both of the proteins, as well as to accommodate automation ofthe assay. Binding of HDx to the target polypeptide, in the presence andabsence of a candidate agent, can be accomplished in any vessel suitablefor containing the reactants. Examples include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows the protein to be boundto a matrix. For example, glutathione-S-transferase/HDx (GST/HDx) fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the cell lysates, e.g. an ³⁵S-labeled, andthe test compound, and the mixture incubated under conditions conduciveto complex formation, e.g. at physiological conditions for salt and pH,though slightly more stringent conditions may be desired. Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel determined directly (e.g. beads placedin scintillant), or in the supernatant after the complexes aresubsequently dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofHDx-binding protein found in the bead fraction quantitated from the gelusing standard electrophoretic techniques such as described in theappended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either HDx ortarget polypeptide can be immobilized utilizing conjugation of biotinand streptavidin. For instance, biotinylated HDx molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive with HDx,but which do not interfere with the interaction between the HDx andtarget polypeptide, can be derivatized to the wells of the plate, andHDx trapped in the wells by antibody conjugation. As above, preparationsof an target polypeptide and a test compound are incubated in theHDx-presenting wells of the plate, and the amount of complex trapped inthe well can be quantitated. Exemplary methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the target polypeptide, or which are reactive with HDxprotein and compete with the target polypeptide; as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the target polypeptide, either intrinsic or extrinsicactivity. In the instance of the latter, the enzyme can be chemicallyconjugated or provided as a fusion protein with the target polypeptide.To illustrate, the target polypeptide can be chemically cross-linked orgenetically fused with horseradish peroxidase, and the amount ofpolypeptide trapped in the complex can be assessed with a chromogenicsubstrate of the enzyme, e.g. 3,3′-diamino-benzadine terahydrochlorideor 4-chloro-1-napthol Likewise, a fusion protein comprising thepolypeptide and glutathione-S-transferase can be provided, and complexformation quantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asanti-HDx antibodies, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes, in addition to the HDx sequence, a secondpolypeptide for which antibodies are readily available (e.g. fromcommercial sources). For instance, the GST fusion proteins describedabove can also be used for quantification of binding using antibodiesagainst the GST moiety. Other useful epitope tags include myc-epitopes(e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) whichincludes a 10-residue sequence from c-myc, as well as the pFLAG system(International Biotechnologies, Inc.) or the pEZZ-protein A system(Pharamacia, NJ).

In another embodiment of a drug screening, a two hybrid assay can begenerated with an HDx and HDx-binding protein. Drug dependent inhibitionor potentiation of the interaction can be scored.

Where the HDx proteins themselves, or in complexes with other proteins,are capable of binding DNA and modifying transcription of a gene, atranscriptional based assay using, for example, an transcriptionalregulatory sequences responsive to HDx complexes operably linked to adetectable marker gene.

Furthermore, each of the assay systems set out above can be generated ina “differential” format as set forth above. That is, the assay formatcan provide information regarding specificity as well as potency. Forinstance, side-by-side comparison of a test compound's effect ondifferent HDxs can provide information on selectivity, and permit theidentification of compounds which selectively modulate the bioactivityof only a subset of the HDx family.

Furthermore, inhibitors of the enzymatic activity of each of the subjectHDx proteins can be identified using assays derived from measuring theability of an agent to inhibit catalytic conversion of a substrate bythe subject proteins. For example, the ability of the subject HDxproteins to deacetylate a histone substrate, such as histone H4 (seeexamples), in the presence and absence of a candidate inhibitor, can bedetermined using standard enzymatic assays.

A number of methods have been employed in the art for assaying histonedeacetylase activity, and can be incorporated in the drug screeningassays of the present invention. In preferred embodiments, the assaywill employ a labeled acetyl group linked to appropriate histone lysineresidues as substrates. In other embodiments, a histone substratepeptide can be labeled with a group whose signal is dependent on thesimultaneous presence or absence of an acetyl group, e.g., the label canbe a fluorogenic group whose fluorescence is modulated (either quenchedor potentiated) by the presence of the acetyl moiety. Using standardenzymatic analysis, the ability of a test agent to cause a statisticallysignificant change in substrate conversion by a histone deacetylase canbe measured, and as desirable, inhibition constants, e.g., K_(i) values,can be calculated. The histone substrate can be provided as a purifiedor semi-purified polypeptide or as part of a cell lysate. Likewise, thehistone deacetylase can be provided to the reaction mixture as apurified or semi-purified polypeptide or as a cell lysate. Accordingly,the reaction mixtures of the subject method can range from reconstitutedprotein mixtures derived with purified preparations of histones anddeacetylases, to mixtures of cell lysates, e.g., by admixing baculoviruslysates containing recombinant histones and deacetylases.

In an exemplary embodiment, the histone substrate for the subject assayis provided by isolation of radiolabeled histones from metabolicallylabelled cells. To illustrate, as described by Hay et al. (1983) J BiolChem 258:3726-3734, HeLa cells can be labelled in culture by addition of[³H]acetate (New England Nuclear) to the culture media. The addition ofbutyrate, trapoxin or the like can be used to increase the abundance ofacetylated histones in the cells. Radiolabelled histones can be isolatedfrom the cells by extraction with H_(S)SO₄ (Marushige et al. (1966) JMol Biol 15:160-174). Briefly, cells are homogenized in buffer,centrifuged to isolate a nuclear pellet, the subsequently homogenizednuclear pellet centrifuged through sucrose, and the resulting chromatinpellet extracted by addition of H_(S)SO₄ to yield [³H]acetyl-labelledhistones. In an alternate embodiment, nucleosome preparations containing[³H]acetyl-labelled histones can be isolated from the labelled cells. Asdescribed in the art, nucleosomes can be isolated from cell preparationsby sucrose gradient centrifugation (Hay et al. (1983) J Biol Chem258:3726-3734; and Noll (1967) Nature 215:360-363), and polynucleosomescan be prepared by NaCI precipitation from micrococcal nuclease digestedcells (Hay et al., supra). Similar procedures for isolating labelledhistones from other cells types, including yeast, have been described.See, for example, Alonso et al. (1986) Biochem Biophys Acta 866:161-169;and Kreiger et al. (1974) J Biol Chem 249:332-334. In yet otherembodiments, the histone is generated by recombinant gene expression,and includes an exogenous tag (e.g., an HA epitope, a poly(his) sequenceor the like) which facilitates in purification from cell extracts. Instill other embodiments, whole nuclei can be isolated from metabolicallylabelled cells by micrococcal nuclease digestion (Hay et al., supra)

In still another embodiment, the deacetylase substrate can be providedas an acetylated peptide including a sequence corresponding to thesequence about the specific lysyl residues acetylated on histone, e.g.,a peptidyl portions of the core histones H2A, H2B, H3 or H4. Suchfragments can be produced by cleavage of acetylated histones derivedfrom metabolically labelled cells, e.g., such as by treatment withproteolytic enzymes or cyanogen bromide (Kreiger et al., supra). Inother embodiments, the acetylated peptide can be provided by standardsolid phase synthesis using acetylated lysine residues (Kreiger et al.,supra).

Continuing with the illustrative use of [³H]acetyl-labelled histones,the activity of a histone deacetylase in the subject assays is detectedby measuring release of [³H]acetate by standard scintillant techniques.In a merely illustrative example, a reaction mixture is provided whichcomprises a recombinant HDx protein suspended in buffer, along with asample of [³H]acetyl-labelled histones and (optionally) a test compound.The reaction mixture is maintained at a desired temperature and pH, suchas 22° C. at pH7.8, for several hours, and the reaction terminated byboiling or other form of denaturation. Released [³H]acetate is extractedand counted. For example, the quenched reaction mixture can be acidifiedwith concentrated HCl, and used to create a biphasic mixture with ethylacetate. The resulting 2 phase system is thoroughly mixed, centrifuged,and the ethyl acetate phase collected and counted by standardscintillation methods. Other methods for detecting acetate release willbe easily recognized by those skilled in the art.

In yet another embodiment, the drug screening assay is derived toinclude a whole cell recombinantly expressing one or more of a targetprotein or HDx protein. The ability of a test agent to alter theactivity of the HDx protein can be detected by analysis of therecombinant cell. For example, agonists and antagonists of the HDxbiological activity can by detected by scoring for alterations in growthor differentiation (phenotype) of the cell. General techniques fordetecting each are well known, and will vary with respect to the sourceof the particular reagent cell utilized in any given assay.

For example, quantification of proliferation of cells in the presenceand absence of a candidate agent can be measured with a number oftechniques well known in the art, including simple measurement ofpopulation growth curves. For instance, where the assay involvesproliferation in a liquid medium, turbidimetric techniques (i.e.absorbence/transmittance of light of a given wavelength through thesample) can be utilized. For example, in the instance where the reagentcell is a yeast cell, measurement of absorbence of light at a wavelengthbetween 540 and 600 nm can provide a conveniently fast measure of cellgrowth. Likewise, ability to form colonies in solid medium (e.g. agar)can be used to readily score for proliferation. In other embodiments, anHDx substrate protein, such as a histone, can be provided as a fusionprotein which permits the substrate to be isolated from cell lysates andthe degree of acetylation detected. Each of these techniques aresuitable for high through-put analysis necessary for rapid screening oflarge numbers of candidate agents.

In addition, where the ability of an agent to cause or reverse atransformed phenotype, growth in solid media such as agar can furtheraid in establishing whether a mammalian cell is transformed.

Additionally, visual inspection of the morphology of the reagent cellcan be used to determine whether the biological activity of the targetedHDx protein has been affected by the added agent. To illustrate, theability of an agent to influence an apoptotic phenotype which ismediated in some way by a recombinant HDx protein can be assessed byvisual microscopy. Likewise, the formation of certain cellularstructures as part of differentiation, such as the formation of neuriticprocess, can be visualized under a light microscope.

The nature of the effect of test agent on reagent cell can be assessedby measuring levels of expression of specific genes, e.g., by reversetranscription-PCR. Another method of scoring for effect on Hdx activityis by detecting cell-type specific marker expression throughimmunofluorescent staining. Many such markers are known in the art, andantibodies are readily available. For example, the presence ofchondroitin sulphate proteoglycans as well as type-II collagen arecorrelated with cartilage production in chondrocytes, and each can bedetected by immunostaining. Similarly, the human kidney differentiationantigen gp160, human aminopeptidase A, is a marker of kidney induction,and the cytoskeletal protein troponin I is a marker of heart induction.In yet another embodiment, the alteration of expression of a reportergene construct provided in the reagent cell provides a means ofdetecting the effect on HDx activity. For example, reporter geneconstructs derived using the transcriptional regulatory sequences, e.g.the promoters, for developmentally regulated genes can be used to drivethe expression of a detectable marker, such as a luciferase gene. In anillustrative embodiment, the construct is derived using the promotersequence from a gene expressed in a particular differentiativephenotype.

It is also deemed to be within the scope of this invention that therecombinant HDx cells of the present assay can be generated so as tocomprise heterologous HDx proteins (i.e. cross-species expression). Forexample, HDx proteins from one species can be expressed in the cells ofanother under conditions wherein the heterologous protein is able torescue loss-of-function mutations in the host cell. For example, thereagent cell can be a yeast cell in which a human MDx protein (e.g.exogenously expressed) is the intended target for development of ananti-proliferative agent. To illustrate, the M778 strain, MATa ura3-52trplΔ1 his3-200 leu2-1 trk1Δrpd3Δ::HIS3, described by Vidal et al.(1991) Mol Cell Biol 6317-6327, which lacks a functional endogenous RPD3gene can be transfected with an expression plasmid including a mammalianHDx gene in order to complement the RPD3 loss-of-function. For example,the coding sequence for HD1 can be cloned into a pRS integrative plasmidcontaining a selectable marker (Sikorski et al. (1989) Genetics122:19-27), and resulting construct used to transform the M778 strain.The resulting cells should produce a mammalian HD1 protein which may becapable performing at least some of the functions of the yeast RPD3protein. The HDx transformed yeast cells can be easier to manipulatethan mammalian cells, and can provide access to certain assay formats,such as turbidity detection methods, which may not be obtainable withmammalian cells.

Moreover, the combination of the “mammalianized” strain with the strainM537 (MATa ura3-52 trplΔ1 his3-200 leu2-1 trk1Δ, Vidal et al., supra)can provide an exquisitely sensitive cell-based assay for detectingagent which specifically inhibit, for example, the yeast RPD3deacetylase.

In another aspect, the invention provides compounds useful forinhibition of HDxs. In a preferred embodiment, an HDx inhibitor compoundof the invention can be represented by the formula A-B-C, in which A isa specificity element for selective binding to an HDx, B is a linkerelement, and C is an electrophilic moiety capable of reacting with anucleophilic moiety of an HDx; with the proviso that the compound is notbutyrate, trapoxin, or trichostatin.

In another aspect, the invention provides an affinity matrix for bindingor purifying an HDx. In a preferred embodiment, the affinity matrix canbe represented by the formula S-A-B-C, in which S is a solid orinsoluble support, and A, B, and C are as described above. The solid orinsoluble support S can be any of a variety of supports, many of whichare known in the art, for synthesis of, or immobilization of, compounds,e.g., peptides, benzodiazepines, and the like. For a review ofsolid-supported synthesis, see, e.g., Hodge et al., Polymer-supportedReactions in Organic Synthesis, John Wiley & Sons, New York, 1980. TheHDx inhibitor moiety A-B-C can be bonded directly to the support S, orcan be bonded to the support S through a linking or spacing moiety, asis known in the art.

In another aspect, the invention provides a method of inhibiting an HDx.The method comprises contacting the HDx with a compound capable ofinhibiting HDx activity, under conditions such that HDx activity isinhibited. In preferred embodiments, the compounds can be represented bythe formula A-B-C, in which A, B, and C are as described above; with theproviso that the compound is not butyrate, trapoxin, or trichostatin.

In another aspect, the invention provides a method of purifying an HDx.The method includes contacting a reaction mixture comprising an HDx withan affinity matrix capable of selectively binding to an HDx, andseparating at least one other component of the reaction mixture from theHDx. In a preferred embodiment, the affinity matrix can be representedby the formula S-A-B-C, in which S, A, B, and C are as described above.

In general, the elements A, B, and C of the inhibitor compounds areselected to permit selective binding to, and inhibition of, at least oneHDx. The elements A, B, and C can be selected to provide specificity forparticular HDxs. For example, a series of candidate HDx inhibitorcompounds can be synthesized, e.g., according to the combinatorialmethods described infra, and the library of candidate compounds screenedagainst one or more HDxs to determine the compound or compounds withoptimal activity and specificity for a particular HDx.

Thus, in preferred embodiments, the specificity element A is selectedsuch that the HDx inhibitor compound binds selectively to an HDx. Ingeneral, the specificity element A will be selected according to factorssuch as the binding specificity of the HDx or HDxs to which theinhibitor compound should bind, ease of synthesis, stability in vivo orin vitro, and the like. In certain embodiments, the specificity elementA is a cyclotetrapeptidyl moiety. In another embodiment, A is asubstituted or unsubstituted aryl moiety. In yet another embodiment, Ais a nonaromatic carbocycle. In still another embodiment, A is an aminoacyl moiety (e.g., a natural or non-natural amino acyl moiety). In yetanother embodiment, A is a heterocyclyl moiety.

In preferred embodiments, B is selected from the group consisting ofsubstituted and unsubstituted C₄-C₈ alkylidene, C₄-C₈ alkenylidene,C₄-C₈ alkynylidene, and D-E-F, in which D and F are independently absentor C₂-C₇ alkylidene, C₂-C₇ alkenylidene, or C₂-C₇ alkynylidene, and E isO, S, or NR′, in which R′ is H, lower alkyl, lower alkenyl, loweralkynyl, aralkyl, aryl, or heterocyclyl. The element B should beselected to permit the specificity element A to interact with an HDxsuch that specific binding occurs, while poising the electrophilicmoiety C for reaction with a nucleophilic moiety of the HDx.

In a preferred embodiment, C is an electrophilic moiety that isapproximately isosteric with an N-acetyl group (i.e., C hasapproximately the same steric bulk as an N-acetyl group) In preferredembodiments, the element C is capable of reacting, covalently ornon-covalently, with a nucleophilic moiety of an HDx. In certainpreferred embodiments, the element C is capable of binding (e.g., bychelation) to a metal ion, e.g., a divalent metal ion, e.g., zinc orcalcium. In preferred embodiments, C is selected from the groupconsisting of a, β-epoxyketones, α,β-epoxythioketones,α,β-epoxysulfoxides, hydroxamic acids, α-haloketones, α-halothioketones,α-diazoketones, α-diazothioketones, vinyl epoxides,trifluoromethylketone, trifluoromethylthioketone, enones (e.g., ofketones or thioketones), ynones (e.g., of ketones or thioketones),α,β-aziridinoketones, hydrazones, boronic acids, carboxylates, amides(e.g., —C(O)-amino), sulfones, aldehyde, alkyl halides, epoxides, andthe like.

In accordance with the foregoing, the moieties A, B, and C canillustratively be represented by the formulas depicted in FIG. 6, inwhich R₁ represents one or more substituents selected from the groupconsisting of amino, halogen, alkyl, alkenyl, alkynyl, aryl, aralkyl,heterocyclyl, azido, carboxyl, alkoxycarbonyl, hydroxyl, alkoxy, cyano,trifluoromethyl, and the like; R″ is C₁-C₈ alkylidene, C₂-C₈alkenylidene, or C₂-C₈ alkynylidene; R₅ is hydrogen, alkyl,alkoxycarbonyl, aryloxycarbonyl, alkylsulfonyl, arylsulfonyl or aryl; R₆is hydrogen, alkyl, aryl, alkoxy, aryloxy, halogen, and the like; R′₆ ishydrogen, alkyl, alkenyl, alkynyl, aryl, and the like; R₇ is hydrogen,alkyl, aryl, alkoxy, aryloxy, amino, hydroxylamino, alkoxylamino,halogen, and the like; R₈ is hydrogen, alkyl, halogen, and the like; R₉is hydrogen, alkyl, aryl, hydroxyl, alkoxy, aryloxy, amino, and thelike; X is a good leaving group, e.g., diazo, halogen, a sulfate orsulfonate ester, e.g., a tosylate or mesylate, and the like; and Y is Oor S.

In certain preferred embodiments, an HDx inhibitor compound can berepresented by the formula A-B-C, in which A is selected from the groupconsisting of cycloalkyls, unsubstituted and substituted aryls,heterocyclyls, amino acyls, and cyclotetrapeptides; B is selected fromthe group consisting of substituted and unsubstituted C₄-C₈ alkylidene,C₄-C₈ alkenylidene, C₄-C₈ alkynylidene, C₄-C₈ enyne, and D-E-F, in whichD and F are independently absent or a C-C₇ alkylidene, an C₂-C₇alkenylidene, or an C₂-C₇ alkynylidene, and E is O, S, or NR′, in whichR′ represents H, a lower alkyl, a lower alkenyl, a lower alkynyl, anaralkyl, aryl, or a heterocyclyl; and C is selected from the groupconsisting of

and B(OH)₂ (boronic acid); in which Z represents O, S, or NR₅, and Y,R₅, R′₆, and R₇ are as defined above. In preferred embodiments, R′₆ ishydrogen. In certain preferred embodiments, B is not a C₄-C₈ alkylidene.In preferred embodiments, if B is a C₄-C₈ alkylidene, C is not a boronicacid. In other preferred embodiments, the inhibitor compound is nottrapoxin.

In certain preferred embodiments, an HDx inhibitor compound can berepresented by the formula A-B-C, in which A is selected from the groupconsisting of cycloalkyls, unsubstituted and substituted aryls,heterocyclyls, amino acyls, and cyclotetrapeptides; B is selected fromthe group consisting of substituted and unsubstituted C₄-C₈ alkylidene,C₄-C₈ alkenylidene, C₄-C₈ alkynylidene, C₄-C₈ enyne, and D-E-F, in whichD and F are independently absent or C₁-C₇ alkylidene, C₂-C₇alkenylidene, or C₂-C₇ alkynylidene, and E is O, S, or NR′, in which R′represents H, a lower alkyl, a lower alkenyl, a lower alkynyl, anaralkyl, an aryl, or a heterocyclyl; and C is selected from the groupconsisting of

in which R₉ is as defined above. In preferred embodiments, B is not aC₄-C₈ alkylidene. In preferred embodiments, the inhibitor compound isnot trichostatin.

In still another preferred embodiment, an HDx inhibitor compound can berepresented by the formula A-B-C, in which A is selected from the groupconsisting of cycloalkyls, unsubstituted and substituted aryls,heterocyclyls, amino acyls, and cyclotetrapeptides; B is selected fromthe group consisting of substituted and unsubstituted C₄-C₈ alkylidene,C₄-C₈ alkenylidene, C₄-C₈ alkynylidene, C₄-C₈ enyne, and D-E-F, in whichD and F are independently absent or a C₁-C₇ alkylidene, a C₂-C₇alkenylidene, or a C₂-C₇ alkynylidene, and E is O, S, or NR′, in whichR′ is H, lower alkyl, lower alkenyl, lower alkynyl, aralkyl, aryl, orheterocyclyl; and C is

in which Y is O or S, and R₇ is as defined above.

Certain HDx inhibitor compounds of the present invention may exist inparticular geometric or stereoisomeric forms. For example, amino acidscan contain at least one chiral center. The present inventioncontemplates all such compounds, including cis- and trans-isomers, R-and S-enantiomers, diastereomers, the racemic mixtures thereof, andother mixtures thereof, as falling within the scope of the invention.Additional asymmetric carbon atoms may be present in a substituent suchas an alkyl group. All such isomers, as well as mixtures thereof, areintended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomer. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts can be formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 4-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification and claims is intended to include both “unsubstitutedalkyls” and “substituted alkyls”, the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, carbonyl (such as a carboxylate, alkoxycarbonyl,aryloxycarbonyl, alkylcarbonyl, arylcarbonyl, aldehyde, and the like),thiocarbonyl (such as a thioacid, alkoxycarbonyl, and the like), analkoxyl, unsubstituted amino, mono- or disubstituted amino, amido,amidine, imine, nitro, azido, sulfhydryl, alkylthio, cyano,trifluoromethyl, sulfonato, sulfamoyl, sulfonamido, heterocyclyl,aralkyl, or an aromatic or heteroaromatic moiety. It will be understoodby those skilled in the art that the moieties substituted on thehydrocarbon chain can themselves be substituted, as described above, ifappropriate. Exemplary substituted alkyls are described below.Cycloalkyls can be further substituted with, e.g., alkyls, alkenyls,alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, —CF₃,—CN, and the like.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.The term “enyne” refers to an unsaturated aliphatic moiety having atleast one double bond and one triple bond.

The terms “alkylidene, ” “alkenylidene, ” and “alkynylidene” areart-recognized and refer to moieties corresponding to alkyl, alkenyl,and alkynyl moieties as defined above, but having two valences availablefor bonding.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, phenyl, pyrrolyl, furanyl, thiophenyl,imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridyl,pyrazinyl, pyridazinyl and pyrimidyl, and the like. Those aryl groupshaving heteroatoms in the ring structure may also be referred to as“aryl heterocycles” or “heteroaromatics”. The aromatic ring can besubstituted at one or more ring positions with such substituents asdescribed above, as for example, halogen, azido, alkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino,amido, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “heterocyclyl” or “heterocyclic group” refer to non-aromatic4- to 10-membered ring structures, more preferably 4- to 7-memberedrings, which ring structures include one to four heteroatoms (e.g., O,N, S, P and the like). Heterocyclyl groups include, for example,pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,alkoxycarbonyl, aryloxycarbonyl, carboxyl, silyl, ether, alkylthio,alkylsulfonyl, arylsulfonyl, ketone (e.g., —C(O)-alkyl or —C(O)-aryl),aldehyde, heterocyclyl, an aryl or heteroaryl moiety, —CF₃, —CN, or thelike.

Compounds represented by the formula A-B-C, in which A, B, and C havethe values described supra, can be synthesized by standard techniques oforganic synthesis. For example, precursor synthons corresponding to eachof the moieties A, B, and C, or subunits thereof, can be coupled inlinear or convergent syntheses to provide HDx inhibitor compounds, orcompounds readily converted thereto. Syntheses of the HDx inhibitorcompound trichostatin, and related compounds, have been reported; see,e.g., Massa, S. et al. (1990) J Med Chem. 33:2845-49; Mori, K., andKosecki, K. (1988) Tetrahedron 44:6013-20; Koseki, K. and Mori, K.European Patent Application EP 331524 A2; Fleming, I. et al. (1983)Tetrahedron 39:841-46. Analogs of trapoxin have also been synthesized;see, e.g., Yoshida, H. and Sugita, K. (1992) Jpn. J. Cancer Res.83:324-28.

Thus, in an illustrative synthesis, a compound represented by theformula A-B-C, in which A is an phenyl group, while B and C can have avariety of values, can be synthesized as shown below:

According to the Scheme, a functionalized organometallic aryl compound(MX=organometallic moiety; R is any substituent; X is a leaving group,e.g., halogen) (e.g., organotin, boronate, aryllithium, cuprate,Grignard reagent, etc.) is alkylated or acylated to providefunctionalized compounds (e.g., the exemplary compounds 1, 2, or 3 whichcan be further elaborated to provide compounds with a wide variety ofsubstituents and carbon backbones. Other A moieties (e.g., specificityelements) can be obtained by use of appropriate synthons, e.g., bysubstituting vinylorganometallic compounds for the organometallic arylcompound of the Scheme (followed by further treatment, e.g., reduction,of the vinyl group, if desired, to yield an alkyl A moiety). By way ofillustration, as shown for compound 1, the carbonyl group can be usedfor elaboration, e.g., by reduction of the carbonyl group to an alcohol,conversion of the alcohol to a tosylate, and nucleophilic displacementof the tosylate by an acyl compound (e.g., a ketone or ester) to providea chain-lengthened product (Route A), which can be converted to a C(O)Xfunctionality (e.g., by hydrolysis of an ester and conversion of theresulting carboxylic acid to an acid chloride). Alternatively, thecarbonyl group of 1 can be used for olefination (Route B), e.g.,Homer-Emmons olefination, to provide an elaborated alkenyl compound.Also, the carbonyl group can be converted to an alkynyl functionality,e.g., via the Corey-Fuchs procedure, to provide an elaborated alkynylcompound. For purposes of clarity, only certain chain lengths andfunctional group patterns are shown in the scheme; however, the skilledartisan will appreciate that many other compounds, with a variety of Bmoieties (i.e., linking moieties), can be synthesized through analogousprocedures. The C(O)X functionality (e.g., an acid chloride where X isCl) can be converted to functional groups such as amide, hydrazido,trifluormethylketone, enone, epoxide, aziridine, and the like, throughmethods conventional in the art. Thus, the synthetic pathways shown inthe Scheme provide access to compounds having a variety of C moieties(e.g., reactive moieties) suitable for substitution in the subject HDxinhibitors.

In vitro chemical synthesis provides a method for generating librariesof compounds that can be screened for ability to bind to or inhibit atarget protein, e.g., an HDx. Although in vitro methods have previouslybeen used in the pharmaceutical industry to identify potential drugs,recently developed methods have focused on rapidly and efficientlygenerating and screening large numbers of compounds and are amenable togenerating HDx inhibitor compound libraries for use in the subjectmethod. The various approaches to simultaneous preparation and analysisof large numbers of compounds (herein “combinatorial synthesis”) eachrely on the fundamental concept of synthesis on a solid supportintroduced for peptides by Merrifield in 1963 (Merrifield, R. B. (1963)J Am Chem Soc 85:2149-2154). Many types of solid matrices have beensuccessfully used in solid-phase synthesis, and can be selectedaccording to the type of chemistry to be performed on the immobilizedmoieties, as is discussed in more detail below.

Several synthetic schemes have been suggested or employed for thecombinatorial synthesis of organic compounds (see, e.g., E. M. Gordon etal., J. Med. Chem. 37:1385-1401 (1994)).

Multipin Synthesis

One method for combinatorial synthesis of compounds is the multipinsynthesis method. Briefly, Geysen and co-workers (Geysen et al. (1984)PNAS 81:3998-4002) introduced a method for generating compounds by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. In the original experiments, about 50nmol of a single compound was covalently linked to the spherical head ofeach pin, and interactions of each compound with a receptor or antibodycould be determined in a direct binding assay. The Geysen technique canbe used to synthesize and screen thousands of compounds per week usingthe multipin method, and the tethered compounds may be reused in manyassays. In subsequent work, the level of compound loading on individualpins has been increased to as much as 2 μmol/pin by grafting greateramounts of functionalized acrylate derivatives to detachable pin heads,and the size of the compound library has been increased (Valerio et al.(1993) Int J Pept Protein Res 42:1-9). Appropriate linker moieties havealso been appended to the pins so that the compounds may be cleaved fromthe supports after synthesis for assessment of purity and evaluation incompetition binding or functional bioassays (Bray et al. (1990)Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

More recent applications of the multipin method have taken advantage ofthe cleavable linker strategy to prepare soluble compound libraries(Maeji et al. (1990) J Immunol Methods 134:23-33; Gammon et al. (1991) JExp Med 173:609-617; Mutch et al. (1991) Pept Res 4:132-137).

Divide-Couple-Recombine

In another embodiment, a variegated library of HDx inhibitor compoundsis provided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135;and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971). Briefly, as thename implies, at each synthesis step where degeneracy (e.g., a pluralityof different moieties) is introduced into the library, the beads aredivided into as many separate groups to correspond to the number ofdifferent residues (e.g., functional groups or other moieties) to beadded at that position, the different residues coupled in separatereactions, and the beads recombined into one pool for the next step.

In one embodiment, the divide-couple-recombine strategy can be carriedout using the so-called “tea bag” method first developed by Houghten,where synthesis occurs on resin that is sealed inside porouspolypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Residuesare coupled to the resins by placing the bags in solutions of theappropriate individual activated monomers, while all common steps suchas resin washing and deprotection (if appropriate) are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound, and the compounds may be liberated fromthe resins using a multiple cleavage apparatus (Houghten et al. (1986)Int J Pept Protein Res 27:673-678). This technique offers advantages ofconsiderable synthetic flexibility and has been partially automated(Beck-Sickinger et al. (1991) Pept Res 4:88-94). Moreover, compounds canbe produced in sufficient quantities (>500 μmol) for purification andcomplete characterization if desired.

Synthesis using the tea-bag approach is useful for the production of alibrary, albeit of limited size, as is illustrated by its use in a rangeof molecular recognition problems including antibody epitope analysis(Houghten et al. (1986) PNAS 82:5131-5135), peptide hormonestructure-function studies (Beck-Sickinger et al. (1990) Int J PeptProtein Res 36:522-530; Beck-Sickinger et al. (1990) Eur J Biochem194:449-456), and protein conformational mapping (Zimmerman et al.(1991) Eur J Biochem 200:519-528).

Combinatorial Synthesis on Nontraditional Solid Supports

The search for innovative methods of solid-phase synthesis has led tothe investigation of alternative polymeric supports to thepolystyrene-divinylbenzene matrix originally popularized by Merrifield.Cellulose, either in the form of paper disks (Blankemeyer-Menge et al.(1988) Tetrahedron Lett 29-5871-5874; Frank et al. (1988) Tetrahedron44:6031-6040; Eichler et al. (1989) Collect Czech Chem Commun54:1746-1752; Frank, R. (1993) Bioorg Med Chem Lett 3:425430) or cottonfragments (Eichler et al. (1991) Pept Res 4:296-307; Schmidt et al.(1993) Bioorg Med Chem Lett 3:441446) has been successfullyfunctionalized for peptide synthesis. Typical loadings attained withcellulose paper range from 1 to 3 μmol/cm², and HPLC analysis ofmaterial cleaved from these supports indicates a reasonable quality forthe synthesized peptides. Alternatively, peptides may be synthesized oncellulose sheets via non-cleavable linkers and then used in ELISA-basedbinding studies (Frank, R. (1992) Tetrahedron 48:9217-9232). The porous,polar nature of this support may help suppress unwanted nonspecificprotein binding effects. In one convenient configuration synthesisoccurs in an 8×12 microtiter plate format. Frank has used this techniqueto map the dominant epitopes of an antiserum raised against a humancytomegalovirus protein, following the overlapping peptide screening(Pepscan) strategy of Geysen (Frank, R. (1992) Tetrahedron48:9217-9232). Other membrane-like supports that may be used forsolid-phase synthesis include polystyrene-grafted polyethylene films(Berg et al. (1989) J Am Chem Soc 111:8024-8026).

Combinatorial Libraries by Light-Directed, Spatially AddressableParallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compoundis given by its locations on a synthesis substrate is termed aspatially-addressable synthesis. In one embodiment, the combinatorialprocess is carried out by controlling the addition of a chemical reagentto specific locations on a solid support (Dower et al. (1991) Annu RepMed Chem 26:271-280; Fodor, S. P. A. (1991) Science 251:767; Pirrung etal. (1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) TrendsBiotechnol 12:19-26). The technique combines two well-developedtechnologies: solid-phase synthesis chemistry and photolithography. Thehigh coupling yields of solid-phase reactions allows efficient compoundsynthesis, and the spatial resolution of photolithography affordsminiaturization. The merging of these two technologies is done throughthe use of photolabile protecting groups, e.g., amino protecting groups,in the synthetic procedure.

The key points of this technology are illustrated in Gallop et al.(1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared forcompound synthesis through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers. Light is usedto selectively activate a specified region of the synthesis support forcoupling. Removal of the photolabile protecting groups by lights(deprotection) results in activation of selected areas. Afteractivation, the first of a set of residues, each bearing a photolabileprotecting group, is exposed to the entire surface. Coupling only occursin regions that were addressed by light in the preceding step. Thereagent solution is removed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed. The target can be labeled with a fluorescent reportergroup to facilitate the identification of specific interactions withindividual members of the matrix.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized in the same number of steps; this leads to thegeneration of many different masking strategies.

Encoded Combinatorial Libraries

In yet another embodiment, the subject method provides an HDx inhibitorcompound library provided with an encoded tagging system. A recentimprovement in the identification of active compounds from combinatoriallibraries employs chemical indexing systems using tags that uniquelyencode the reaction steps a given bead has undergone and, by inference,the structure it carries. Conceptually, this approach mimics phagedisplay libraries, where activity derives from expressed peptides, butthe structures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. Two forms of encoding have beenreported: encoding with sequenceable bio-oligomers (e.g.,oligonucleotides and peptides), and binary encoding withnon-sequenceable tags.

Tagging with Sequenceable Bio-oligomers

The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89:5381-5383), and an example of such a library appeared the followingyear (Needles et al. (1993) PNAS 90:10700-10704). A combinatoriallibrary of nominally 7⁷ (=823,543) peptides composed of all combinationsof Arg, Gin, Phe, Lys, Val, D-Val and Thr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected NH₂groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-mers, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following such techniques, HDx inhibitor compoundlibraries can be derived and screened using HDxs of the subjectinvention.

It is noted that an alternative approach useful for generatingnucleotide-encoded synthetic peptide libraries employs a branched linkercontaining selectively protected OH and NH₂ groups (Nielsen et al.(1993) J Am Chem Soc 115:9812-9813; and Nielsen et al. (1994) MethodsCompan Methods Enzymol 6:361-371). This approach requires that equimolarquantities of test peptide and tag co-exist, though this may be apotential complication in assessing biological activity, especially withnucleic acid based targets.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesison non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test HDx inhibitorcompound library member for bioassay, in part (as described infra)because assays employing beads limit the choice of targets, and in partbecause the tags are potentially susceptible to biodegradation.

Peptides themselves have been employed as tagging molecules forcombinatorial libraries. Two exemplary approaches are described in theart, both of which employ branched linkers to solid phase upon whichcoding and ligand strands are alternately elaborated. In the firstapproach (Kerr J M et al. (1993) J Am Chem Soc 115:2529-2531),orthogonality in synthesis is achieved by employing acid-labileprotection for the coding strand and base-labile protection for theligand strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170),branched linkers are employed so that the coding unit and the testpeptide are both attached to the same functional group on the resin. Inone embodiment, a linker can be placed between the branch point and thebead so that cleavage releases a molecule containing both code andligand (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894). In anotherembodiment, the linker can be placed so that the test peptide can beselectively separated from the bead, leaving the code behind. This lastconstruct is particularly valuable because it permits screening of thetest peptide without potential interference, or biodegradation, of thecoding groups. Examples in the art of independent cleavage andsequencing of peptide library members and their corresponding tags hasconfirmed that the tags can accurately predict the peptide structure.

It is noted that peptide tags are more resistant to decomposition duringligand synthesis than are oligonucleotide tags, but they must beemployed in molar ratios nearly equal to those of the ligand on typical130 μm beads in order to be successfully sequenced. As witholigonucleotide encoding, the use of peptides as tags requires complexprotection/deprotection chemistries.

Non-sequenceable Tagging: Binary Encoding

An alternative form of encoding the test peptide library employs a setof non-sequenceable tagging molecules (e.g., molecules havingelectrophoric. moieties) that are used as a binary code (Ohlmeyer et al.(1993) PNAS 90:10922-10926). Exemplary tags are haloaromatic alkylethers that are detectable as their trimethylsilyl ethers at less thanfemtomolar levels by electron capture gas chromatography (ECGC).Variations in the length of the alkyl chain, as well as the nature andposition of the aromatic halide substituents, permit the synthesis of atleast 40 such tags, which in principle can encode 240 (e.g., upwards of10¹²) different molecules. In the original report (Ohlmeyer et al.,supra) the tags were bound to about 1% of the available amine groups ofa peptide library via a photocleavable O-nitrobenzyl linker. Thisapproach is convenient when preparing combinatorial libraries ofpeptides or other amine-containing molecules. A more versatile systemhas, however, been developed that permits encoding of essentially anycombinatorial library. Here, the ligand is attached to the solid supportvia the photocleavable linker and the tag is attached through a catecholether linker via carbene insertion into the bead matrix (Nestler et al.(1994) J Org Chem 59:4723-4724). This orthogonal attachment strategypermits the selective detachment of library members for bioassay insolution and subsequent decoding by ECGC after oxidative detachment ofthe tag sets.

Binary encoding with tags, e.g., electrophoric tags, has beenparticularly useful in defining selective interactions of substrateswith synthetic receptors (Borchardt et al. (1994) J Am Chem Soc116:373-374), and model systems for understanding the binding andcatalysis of biomolecules. Even using detailed molecular modeling, theidentification of the selectivity preferences for synthetic receptorshas required the manual synthesis of dozens of potential substrates. Theuse of encoded libraries makes it possible to rapidly examine all themembers of a potential binding set. The use of binary-encoded librarieshas made the determination of binding selectivities so facile thatstructural selectivity has been reported for four novel syntheticmacrobicyclic and tricyclic receptors in a single communication(Wennemers et al. (1995) J Org Chem 60:1108-1109; and Yoon et al. (1994)Tetrahedron Lett 35:8557-8560) using the encoded library mentionedabove. Similar facility in defining specificity of interaction would beexpected for many other biomolecules.

Although the several amide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the tags arc attached directly to thesolid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031) and provideguidance for generating the subject HDx inhibitor compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: first,multiple beads are placed in 96-well microtiter plates; second, ligandsare partially detached and transferred to assay plates; third, abioassay identifies the active wells; fourth, the corresponding beadsare rearrayed singly into new microtiter plates; fifth, single activecompounds are identified; and sixth, the structures are decoded.

The above approach was employed in screening for carbonic anhydrase (CA)binding and identified compounds which exhibited nanomolar affinitiesfor CA. Unlike sequenceable tagging, a large number of structures can berapidly decoded from binary-encoded libraries (a single ECGC apparatuscan decode 50 structures per day). Thus, binary-encoded libraries can beused for the rapid analysis of structure-activity relationships andoptimization of both potency and selectivity of an active series. Thesynthesis and screening of large unbiased binary encoded HDx inhibitorcompound libraries for lead identification, followed by preparation andanalysis of smaller focused libraries for lead optimization, offers aparticularly powerful approach to discovery of HDx inhibitor compounds.

HDx inhibitor compounds can be synthesized on solid support byappropriate functionalization for attachment to a solid matrix, oralternatively, by solution-phase synthesis followed by immobilaizationthrough an appropriate functional group. Thus, in an illustrativeembodiment, an HDx inhibitor compound, which is analogous totrichostatin, can be synthesized on a solid support by attachmentthrough an amino group of the specificity element A, as shown in FIG. 7.The solid support is preferably capable of withstanding syntheticconditions required to synthesize the requisite compounds. The compoundcan preferably be released from the solid support, e.g., by selectivecleavage of an amide bond.

The synthetic steps employed to synthesize compounds on solid supportare preferably selected to allow a wide variety of residues (e.g.,building blocks) to be coupled to the immobilized moieties, preferablyunder mild conditions. Suitable reaction chemistries include well-knowncarbon-carbon bond forming reactions such as the Stille and Suzukicouplings, as well as Horner-Emmons reactions, Ni/Cr mediated couplings,and the like. Particularly preferred coupling reactions can be performedin the presence of water and do not require harsh conditions orexpensive reagents.

Thus, in an exemplary synthesis shown in FIG. 7, substitutedN-methyl4-(tributyltin)anilines (in which R₁ represents one or moresubstitutions, e.g., hydrogen, halogen, alkyl, alkoxy, and the like) arecoupled in a plurality of reaction vessels to beads of a solid support(e.g., Affigel). The beads are further divided into a plurality ofreaction vessels, and suspended in a solvent such as DMF, and one acidchloride building block (corresponding to linking element B) isintroduced into each vessel (R₂ and R₃ represent, e.g., hydrogen,halogen, alkyl, and the like; and the broken line represents an optionaldouble bond). The reactions are stirred under an inert gas (e.g.nitrogen) and a palladium catalyst (e.g., Pd(PPh₃)₄) is added (0.1-1.0mol %). The reaction is stirred for 1-24 hours. Upon completion of thereaction, the beads are washed, and placed in a plurality of vessels.The aldehyde moiety is deprotected by mild acid treatment (e.g., PPTS inMeOH), and the beads are again washed and placed in a plurality ofreaction vessels, and the beads are suspended in dry acetonitrile. Onebuilding block (corresponding to the reactive element C) is then addedto each reaction vessel. As illustratively shown in FIG. 7, a pluralityof phosphonates can be employed (R₄ represents, e.g., alkyl, alkenyl,alkynyl, alkoxy, and the like). A Homer-Emmons reaction is performed byaddition of LiCl (1.1 equiv.) and diisopropylethylamine (DIPEA) or DBU(1.2 equiv). Upon completion of the reaction, the beads are washed withwater and acetonitrile, and then dried to yield a library of candidateHDx inhibitor compounds on solid support. The compounds can then bereleased from the solid support into solution; or the compounds can bescreened while attached to the solid support.

The above combinatorial synthesis can be performed in an encoded mode,e.g., the binary tagging method described supra, by addition of theappropriate tag for each monomer. In this mode, after each reaction hasbeen performed and the corresponding tag attached, the beads from allreactions can be recombined and then divided into aliquots for furtherderivatization. This method provides the advantage of ease of handlingwhen large libraries are to be synthesized. Regardless of the method ofsynthesis, the combinatorial library can be screened for activityaccording to known methods (see, e.g., Gordon et al., supra).

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin; or (4) intravaginally or intrarectally,for example, as a pessary, cream or foam.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a deacetylaseinhibitor of the present invention which is effective for producing somedesired therapeutic effect by inhibiting histone deacetylation in atleast a sub-population of cells in an animal and thereby blocking thebiological consequences of that event in the treated cells, at areasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject deacetylaseinhibitor agent from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present deacetylaseinhibitors may contain a basic functional group, such as amino oralkylamino, and are, thus, capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptableacids. The term “pharmaceutically-acceptable salts” in this respect,refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or by separately reacting a purified compound of theinvention in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

In other cases, the deacetylase inhibitory compounds of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of compounds of the present invention.These salts can likewise be prepared in situ during the final isolationand purification of the compounds, or by separately reacting thepurified compound in its free acid form with a suitable base, such asthe hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, with ammonia, or with a pharmaceutically-acceptableorganic primary, secondary or tertiary amine. Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like. (See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the deacetylase inhibitor which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a deacetylase inhibitor of the presentinvention with liquid carriers, or finely divided solid carriers, orboth, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A deacetylase inhibitor ofthe present invention may also be administered as a bolus, electuary orpaste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium. phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered deacetylaseinhibitor moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the deacetylaseinhibitors of the invention include pharmaceutically acceptableemulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, the liquid dosage forms maycontain inert diluents commonly used in the art, such as, for example,water or other solvents, solubilizing agents and emulsifiers, such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active deacetylase inhibitor, maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active deacetylase inhibitor.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of adeacetylase inhibitor of this invention include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive deacetylase inhibitor of this invention, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the deacetylase inhibitorin the proper medium. Absorption enhancers can also be used to increasethe flux of the deacetylase inhibitor across the skin. The rate of suchflux can be controlled by either providing a rate controlling membraneor dispersing the deacetylase inhibitor in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more deacetylase inhibitors of theinvention in combination with one or more pharmaceutically-acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject deacetylase inhibitors in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred.

These deacetylase inhibitor may be administered to humans and otheranimals, for therapy by any suitable route. of administration, includingorally, nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular deacetylase inhibitor employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular deacetylaseinhibitor employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

Another aspect of the present invention relates to a method of inducingand/or maintaining a differentiated state, enhancing survival, and/orinhibiting (or alternatively potentiating) proliferation of a cell, bycontacting the cells with an agent which modulates HDx-dependenttranscription. For instance, it is contemplated by the invention that,in light of the present finding of an apparently broad involvement ofHDx proteins in the control of chromatin structure and, thus,transcription and replication, the subject method could be used togenerate and/or maintain an array of different tissue both in vitro andin vivo. An “HDx therapeutic,” whether inhibitory or potentiating withrespect to modulating histone deacetylation, can be, as appropriate, anyof the preparations described above, including isolated polypeptides,gene therapy constructs, antisense molecules, peptidomimetics or agentsidentified in the drug assays provided herein.

The HDx compounds of the present invention are likely to play animportant role in the modulation of cellular proliferation. There are awide variety of pathological cell proliferative conditions for which HDxtherapeutics of the present invention may be. used in treatment. Forinstance, such agents can provide therapeutic benefits where the generalstrategy being the inhibition of an anomalous cell proliferation.Diseases that might benefit from this methodology include, but are notlimited to various cancers and leukemias, psoriasis, bone diseases,fibroproliferative disorders such as involving connective tissues,atherosclerosis and other smooth muscle proliferative disorders, as wellas chronic inflammation.

In addition to proliferative disorders, the present inventioncontemplates the use of HDx therapeutics for the treatment ofdifferentiative disorders which result from, for example,de-differentiation of tissue which may (optionally) be accompanied byabortive reentry into mitosis, e.g. apoptosis. Such degenerativedisorders include chronic neurodegenerative diseases of the nervoussystem, including Alzheimer's disease, Parkinson's disease, Huntington'schorea, amylotrophic lateral sclerosis and the like, as well asspinocerebellar degenerations. Other differentiative disorders include,for example, disorders associated with connective tissue, such as mayoccur due to de-differentiation of chondrocytes or osteocytes, as wellas vascular disorders which involve de-differentiation of endothelialtissue and smooth muscle cells, gastric ulcers characterized bydegenerative changes in glandular cells, and renal conditions marked byfailure to differentiate, e.g. Wilm's tumors.

It will also be apparent that, by transient use of modulators of HDxactivities, in vivo reformation of tissue can be accomplished, e.g. inthe development and maintenance of organs. By controlling theproliferative and differentiative potential for different cells, thesubject HDx therapeutics can be used to reform injured tissue, or toimprove grafting and morphology of transplanted tissue. For instance,HDx antagonists and agonists can be employed in a differential manner toregulate different stages of organ repair after physical, chemical orpathological insult. For example, such regimens can be utilized inrepair of cartilage, increasing bone density, liver repair subsequent toa partial hepatectomy, or to promote regeneration of lung tissue in thetreatment of emphysema. The present method is also applicable to cellculture techniques.

In one embodiment, the HDx therapeutic of the present invention can beused to induce differentiation of uncommitted progenitor cells andthereby give rise to a committed progenitor cell, or to cause furtherrestriction of the developmental fate of a committed progenitor celltowards becoming a terminally-differentiated cell. For example, thepresent method can be used in vitro or in vivo to induce and/or maintainthe differentiation of hematopoietic cells into erythrocytes and othercells of the hematopoietic system. In an illustrative embodiment, theeffect of erythropioetin (EPO) on the growth of EPO-responsive erythroidprecursor cells is increased to influence thier differentiation into redblood cells. For example, as a result of administering an inhibitor ofhistone deacetylation, the amount of EPO, or other diferentiating agent,required for growth and/or differentiation is reduced (PCT/US92/07737).Accordingly, the HDx therapeutics of the present invention, particularlythose which antagonize HDx deacetylase activity, can be administeredalone or in conjunction with EPO and in a suitable carrier tovertebrates to promote erythropoiesis. Alternatively, cells could betreated ex vivo. Such treatment is contemplated in the treatment of avariety of disease states, including in individuals who require bonemarrow transplants (e.g. patients with aplastic anemia, acute leukemias,recurrent lymphomas, or solid tumors).

To illustrate, prior to receiving a bone marrow transplant, a recipientis prepared by ablating or removing endogenous hematopoietic stem cells.Such treatment is usually carried out by total body irradiation ordelivery of a high dose of an alkylating. agent or otherchemotherapeutic, cytotoxic agent, Anklesaria, et al. (1987) PNAS84:7681-7685). Following preparation of the recipient, donor bone marrowcells are injected intravenously. Optionally, the HDx therapeutics ofthe present invention could be contacted with the cells ex vivo oradministered to the subject with the reimplanted cells.

It is also contemplated that there may be cell-type specific HDxproteins, and/or that some cell types may be more sensitive tomodulation of HDx deacetylase activities. Even within a cell type, thestage of differentiation or position in the cell cycle could influencetheir response to an HDx therapeutic. Accordingly, the present inventioncontemplates the use of agents which modulate histone deacetylaseactivity to specificly inhibit or activate certain cell types. In anillustrative example, T cell proliferation could be preferentiallyinhibited in order to induce tolerence by using a procedure similar tothat for inducing tolerance using sodium butyrate (see, for example,PCT/US93/03045). To illustrate, the HDx therapeutics of the presentinvention may be used to induce antigen-specific tolerance in anysituation in which it is desirable to induce tolerence, such asautoimmune diseases, in allogeneic or xenogeneic transplant recipients,or in graft versus host (GVH) reactions. According to the invention,tolerence will typically be induced by presenting the tolerizingcompound (e.g., an HDx inhibitor) substantially contemporaneously withthe antigen, i.e. reasonably close together in time with the antigen. Inpreferred embodiments the HDx therapeutic will be administered afterpresentation of the antigen, so that they will have their effect afterthe particular repertoire of Th cells begins to undergo clonalexpansion.

Yet another aspect of the present invention concerns the application ofHDx therapeutics to modulating morphogenic signals involved inorganogenic pathways. Thus, it is contemplated by the invention thatcompositions comprising HDx therapeutics can also be utilized for bothcell culture and therapeutic methods involving generation andmaintenance of tissue.

In a further embodiment of the invention, the subject HDx therapeuticswill be useful in increasing the amount of protein produced by a cell orrecombinant cell. The cell may include any primary cell isolated fromany animal, cultured cells, immortalized cells, and established celllines. The animal cells used in the present invention include cellswhich intrinsically have an ability to produce a desired protein; cellswhich are induced to have an ability to produce a desired protein, forexample, by stimulation with a cytokine such as an interferon, aninterleukin; genetically engineered cells into which a gene for adesired protein is introduced. The protein produced by the process couldinclude any peptides or proteins, including peptide hormone orproteinaceous hormones such as any useful hormone, cytokine,interleukin, or protein which it may be desirable to have in purifiedform and/or in large quantity.

Another aspect of the invention features transgenic non-human animalswhich express a heterologous HDx gene of the present invention, or whichhave had one or more genomic HDx genes disrupted in at least one of thetissue or cell-types of the animal. Accordingly, the invention featuresan animal model for developmental diseases, which animal has one or moreHDx allele which is mis-expressed. For example, a mouse can be bredwhich has one or more HDx alleles deleted or otherwise renderedinactive. Such a mouse model can then be used to study disorders arisingfrom mis-expressed HDx genes, as well as for evaluating potentialtherapies for similar disorders.

Another aspect of the present invention concerns transgenic animalswhich are comprised of cells (of that animal) which contain a transgeneof the present invention and which preferably (though optionally)express an exogenous HDx protein in one or more cells in the animal. AnHDx transgene can encode the wild-type form of the protein, or canencode homologs thereof, including both agonists and antagonists, aswell as antisense constructs. In preferred embodiments, the expressionof the transgene is restricted to specific subsets of cells, tissues ordevelopmental stages utilizing, for example, cis-acting sequences thatcontrol expression in the desired pattern. In the present invention,such mosaic expression of an HDx protein can be essential for many formsof lineage analysis and can additionally provide a means to assess theeffects of, for example, lack of HDx expression which might grosslyalter development in small patches of tissue within an otherwise normalembryo. Toward this and, tissue-specific regulatory sequences andconditional regulatory sequences can be used to control expression ofthe transgene in certain spatial patterns. Moreover, temporal patternsof expression can be provided by, for example, conditional recombinationsystems or prokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that.catalyzes the genetic recombination a target sequence. As used herein,the phrase “target sequence” refers to a nucleotide sequence that isgenetically recombined by a recombinase. The target sequence is flankedby recombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of one of the subject HDx proteins. For example, excision ofa target sequence which interferes with the expression of a recombinantHDx gene, such as one which encodes an antagonistic homolog or anantisense transcript, can be designed to activate expression of thatgene. This interference with expression of the protein can result from avariety of mechanisms, such as spatial separation of the HDx gene fromthe promoter element or an internal stop codon. Moreover, the transgenecan be made wherein the coding sequence of the gene is flanked byrecombinase recognition sequences and is initially transfected intocells in a 3′ to 5′ orientation with respect to the promoter element. Insuch an instance, inversion of the target sequence will reorient thesubject gene by placing the 5′ end of the coding sequence in anorientation with respect to the promoter element which allow forpromoter driven transcriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems. Cre recombinase catalyzes thesite-specific recombination of an intervening target sequence locatedbetween loxP sequences. loxP sequences are 34 base pair nucleotiderepeat sequences to which the Cre recombinase binds and are required forCre recombinase mediated genetic recombination. The orientation of loxPsequences determines whether the intervening target sequence is excisedor inverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of a recombinant HDx protein can be regulatedvia control of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant HDx protein requires the construction of a transgenic animalcontaining transgenes encoding both the Cre recombinase and the subjectprotein. Animals containing both the Cre recombinase and a recombinantHDx gene can be provided through the construction of “double” transgenicanimals. A convenient method for providing such animals is to mate twotransgenic animals each containing a transgene, e.g., an HDx gene andrecombinase gene.

One advantage derived from initially constructing transgenic animalscontaining an HDx transgene in a recombinase-mediated expressible formatderives from the likelihood that the subject protein, whether agonisticor antagonistic, can be deleterious upon expression in the transgenicanimal. In such an instance, a founder population, in which the subjecttransgene is silent in all tissues, can be propagated and maintained.Individuals of this founder population can be crossed with animalsexpressing the recombinase in, for example, one or more tissues and/or adesired temporal pattern. Thus, the. creation of a founder population inwhich, for example, an antagonistic HDx transgene is silent will allowthe study of progeny from that founder in which disruption of HDxmediated induction in a particular tissue or at certain developmentalstages would result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the HDx transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

Moreover, expression of the conditional transgenes can be induced bygene therapy-like methods wherein a gene encoding the trans-activatingprotein, e.g. a recombinase or a prokaryotic protein, is delivered tothe tissue and caused to be expressed, such as in a cell-type specificmanner. By this method, an HDx transgene could remain silent intoadulthood until “turned on” by the introduction of the trans-activator.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonic target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonic target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). Asa consequence, all cells of the transgenic non-human animal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. Microinjection ofzygotes is the preferred method for incorporating transgenes inpracticing the invention.

Retroviral infection can also be used to introduce HDx transgenes into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonicstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

Methods of making HDx knock-out or disruption transgenic animals arealso generally known. See, for example, Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert recombinase target sequences flankingportions of an endogenous HDx gene, such that tissue specific and/ortemporal control of inactivation of an HDx allele can be controlled asabove.

Exemplification

The invention, now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention and are not intended to limit the invention.

EXAMPLE 1

Trapoxin is a microbially derived cyclotetrapeptide that inhibitshistone deacetylation in vivo and causes mammalian cells to arrest inthe cell cycle. A trapoxin affinity matrix was used to isolate twonuclear proteins that copurified with histone deacetylase activity. Bothproteins were identified by peptide microsequencing, and a cDNA encodingthe histone deacetylase catalytic subunit (HD1) was cloned from a JurkatT cell library. As the predicted protein is highly similar to the yeasttranscriptional regulator RPD3, this study supports a role for histonedeacetylase as a key regulator of eukaryotic transcription.

A requirement for a functional histone deacetylase in cell cycleprogression has been implicated by the discovery that two cytostaticagents, trapoxin and trichostatin (FIG. 1A), inhibit histonedeacetylation in cultured mammalian cells and in fractioned cellextracts (4). In addition to causing G₁ and G₂ phase cell cycle arrest,these natural products alter gene expression and induce certainmammalian cell lines to differentiate. Whereas sodium butyrate also hasthese properties, both trapoxin and trichostatin are five orders ofmagnitude more potent.

Trapoxin is an “irreversible” inhibitor of histone deacetylase activityand its molecular structure offers clues as to how it could form acovalent bond with a nucleophilic active site residue. First, trapoxincontains an electrophilic epoxyketone that is essential for biologicalactivity (5). Second, the aliphatic epoxyketone side chain isapproximately isosteric with N-acetyl lysine (FIG. 1A). Trapoxin likelyacts as a substrate mimic, with epoxyketone poised to alkylate an activesite nucleophile. We therefore regarded trapoxin as a tool that couldreveal the molecular identity of histone deacetylase, so that its rolein transcriptional regulation and cell cycle progression could beelucidated.

Tritium-labeled trapoxin was prepared by total synthesis and used toidentify trapoxin binding protein in crude extracts from bovine thymus.We used a charcoal precipitation assay to detect a specific trapoxinbinding activity primarily in the nuclear fraction of the extracts (6).The binding activity was saturable with nanomolar concentrations. of[³H]trapoxin and was completed by the simultaneous addition of unlabledtrapoxin. Trichostatin also competed with [³H]trapoxin (for synthesis,see Example 2), suggesting that both of these compounds exert theircellular effects by targeting the same molecule.

If trapoxin and trichostatin induce cell cycle arrest by directlyinhibiting histone deacetylase, then the binding and enzymaticactivities should copurify. To investigate this possibility, wefractioned nuclear thymus proteins by ammonium sulfate precipitation andMono Q anion exchange chromatography.

Briefly, thymocytes (˜12 g) prepared from fresh bovine thymus werehomgenized in hypotonic lysis buffer [20 mM tris (pH 7.8), 20 mM NaCl, 1mM EDTA, 10% glycerol, 1 mM PMSF, ImM benzamidine, 10 μg/ml each ofpepstatin, aprotinin, and leupeptin] by mechanical disruption and thenuclei were isolated by centrifugation at 3000 g. Nuclei wereresuspended in lysis buffer and the proteins were extracted with 0.4 Mammonium sulfate. The viscous lysate was sonicated and clarified bycentrifugation at 100,000 g for one hour. Proteins were thenprecipitated with 90% saturated ammonium sulfate and recovered bycentrifugation (100,000 g, one hour). After through dialysis against Qbuffer (25 mM tris pH 8, 10 mM NH Cl, 0.25 mM EDTA, 10% glycerol), aportion of the nuclear proteins (˜12 mg total protein) was loaded onto aHR 10/10 Mono Q column (Pharmacia). The column was washed with 25 ml Qbuffer and eluted with a 50 ml linear gradient of 10 to 500 mM NH₄Cl.The column was further washed with 25 ml 500 mM NH₄ and 25 ml 1 Mhistone deacetylase activities or further purified with the K-trapaffinity matrix. All procedures were done at 40° C.

Two peaks of histone deactylase activity eluted from the Mono Q columnbetween 250 and 350 mM NH₄Cl (FIG. 1B). Trapoxin binding activity, asrevealed by the charcoal precipitation assay (40 nM [³H]trapoxin),precisely coeluted with the histone deacetylase peaks. Furthermore, alldetectable histone deacetylase activity was abolished by treatment witheither trapoxin or trichostatin (20 nM). Similar results were obtainedwith Mono Q fractioned nuclear extracts prepared form human Jurkat Tcells.

To purify the histone deacetylase further, we synthesized an affinitymatrix based on the trapoxin structure. Because trapoxin itself is notamenable to derivatization and the epoxyketone side chain isindispensable for activity, we chose to replace one of the phenylalanineresidues of trapoxin's cyclic core with a lysine that could then becovalently linked to a solid support. This molecule, which we callK-trap, was prepared by a twenty step synthesis starting withcommercially available (R)-proline and (S,S)-threiitol acetonide (FIG.2A) (see Example 3). Synthetic K-trap inhibited [³H]thymidineincorporation in MG-63 human osteosarcoma cells with a potencyapproximately one tenth that of trapoxin. In vitro histone deacetylaseactivity was also inhibited potently by this compound (completeinactivation at 20 nM) (8).

K-trap was deprotected with Pd(Ph₃P)₄ and coupled to an activatedagarose matrix (FIG. 2A). Mono Q fractions containing nuclear proteinsfrom bovine thymus were incubated with the K-trap affinity matrix andthen tested for both trapoxin binding and histone deacetylase activity.Both activities were depleted (90%) by treatment with the K-trap matrix,yet a control matrix capped with ethanolamine had no effect on eitheractivity (8). Bound polypeptides were eluted by boiling the matrix in 1%SDS buffer and separated b polyacrylamide gel electrophoresis. In vitrobinding experiments with soluble [³H]trapoxin indicated that theradiolabel is released into solution following protein denaturation withSDS or gunaidinium hydrochloride. Thus, trapoxin binding proteins wereexpected to elute from the affinity matrix with SDS.

The silver stained gel of the affinity matrix eluates revealed six majorpolypeptides with apparent molecular sizes between 45 and 50 kD (FIG.2B). The interaction between bovine p46-p50 and the K-trap matrixappeared to be specific, because these proteins were not retained whenthe incubation was done in the presence of either trapoxin ortrichostatin (FIG. 2B), nor were they structurally unrelated histonedeacetylase inhibitor, trichostatin, to prevent p46-p50 from binding tothe K-trap matrix implies that one or more of these polypeptidesconstitute the biologically relevant protein target of both trapoxin andtrichostatin. When the affinity purification was repeated with Jurkatnuclear extracts, only two major bands, p50 and p55, were observed bysilver staining (FIG. 2B). Recovery of human p50 and p55 was similarlyabolished by trapoxin (FIG. 2B) and trichostatin (8). Because therelative intensities of bovine p46-p49 vary with each proteinpreparation, we suspect that they are proteolytic fragments derived fromthe bovine equivalent of human p55. One of the bands (p50) is common toboth human and bovine sources.

Large scale purification of the bovine proteins led to the resolution oftwo major bands of ˜46 and ˜50 kD in the final preparativeelectrophoresis step, both of which were submitted for microsequencing.

To obtain enough trapoxin binding protein for microsequencing, nuclearammonium sulfate pellets from 15 bovine thymuses were prepared asdescribed above. Sedimented proteins were resuspended in and dialyzedagainst buffer A [20 mM bistris (pH 7.2), 20 mM NaCl, 10% glycerol] for12 hours, and brought to pH 5. 8 by dialyzing against bugger A (pH 5.8)for 30 minutes. After centrifugation, the dialysate (˜650 mg protein)was loaded onto a Q Sepharose FF column (2.6×10 cm; Pharmacia) and thecolumn was washed with 120 ml buffer A (pH 5.8). Proteins wee elutedwith a 400 ml linear gradient of 20 to 600 mM NaCl in buffer A.Fractions (10 ml; each fraction contained 1 ml of 1 M tris pH 8 toneutralize the acidic buffer A) were assayed for trapoxin bindingactivity. Tween-20 was added to active fractions at a finalconcentration of 0.05%, and these fractions were incubated with K-trapaffinity matrix for 16 hours (25 μl per ml Q fraction). After washingthe matrix three times with phosphate buffered saline, bound proteinswere eluted by boiling in 40 μl of SDS sample buffer per 25 μl ofmatrix. SDS eluates were combined and the proteins resolved by SDS-PVDFmembrane (Biorad). Staining with Ponceau S revealed two major bands (46and 50 kD). The excised bands were proteolytically digested and the HPLCpurified peptide fragments were sequenced at the Harvard MicrochemistryFacility.

The bovine protein of larger molecular size (˜50 kD) corresponds to aknown protein, RbAp48 (11), that consists of seven WD repeat domains(12). Originally identified as a protein that binds to theretinoblastoma gene product (pRb), RbAp48 may constitute an adaptorsubunit that targets the histone deacetylase to specific chromatindomains.

The ˜46 kD bovine protein is highly related to the protein encoded bythe yeast RPD3 gene, which has been implicated by several geneticscreens as a transcriptional regulator, but whose biochemical functionis unknown (13). Partial cDNA sequences for the human gene wereidentified in the expressed sequence tag database (dbEST) and were usedto design polymerase chain reaction (PCR) primers. Briefly, after notingsequence similarity between peptides derived from the purified bovinetrapoxin binding protein and yeast RPD3, we checked dbEST to see whetherany partial sequences for the human homologue had been reported. TwoESTs (Genbank accession numbers: D31480 and F07807) were identifiedwhose predicted translation products aligned with high sequencesimilarity to NH₂— and COOH-terminal regions of HD1, respectively, PCRprimers were designed based on these tags and a one kilobase PCR productwas obtained from a Jurkat cDNA library (Stratagene). A ³²p labeledprobe prepared by random priming was used to screen the Jurkat library,and ten positive clones were isolated. One of the clones was fullysequenced and found to contain a putative full-length open reading frame(FIG. 3A). The peptide sequences obtained from the purified bovineprotein align with 100% identity to sequences deduced from this codingregion (FIG. 3A, boxed residues). We call this human protein HD1 (forhistone deacetylase), and its predicted size of 55 kD agrees well withthe estimated size of p55 isolated from Jurkat nuclear extracts usingthe K-trap affinity matrix (FIG. 2B). A dbEST search indicated theexistence of at least two other related human genes.

To determine the relationship between the proteins from bovine thymus(p46-p50) and the proteins isolated from human Jurkat T cells (p50 andp55), an antiserum was generated against a peptide specified by the HD1open reading frame (FIG. 3A, amino acids 319 to 334). Immunoblotanalysis of the bovine proteins p46-p49 and the human protein p55 showedthat they all react with the antiserum and provides additional evidencethat these bands correspond to bovine and human HD1 (FIG. 3B). Amonoclonal antibody that specifically recognizes RbAp48 was used toconfirm the identity of bovine and hum p50. Importantly, neither HD1 norRbAp48 was detected when the affinity purification was done in thepresence of trapoxin or trichostatin (FIG. 3B).

We used affinity purified antibodies directed against a COOH-terminalpeptide (amino acids 467 to 482) to immunoprecipitate HD1 from crudenuclear extracts. The immnunoprecipitates contained histone deacetylaseactivity that was inhibited by both trapoxin and trichostatin (FIG. 4A).Consistent with the idea that HD1 and RbAp48 form a complex in vivo, thetwo proteins coprecipitated with the anti-DHI antibodies (FIG. 4B).Neither HD1, RbAp48, nor the associated histone deacetylase activitywere immunoprecipitated in the presence of the HD1 COOH-terminal peptide(FIGS. 4A and 4B) (15). HD1, like RbAp48 (11), is detected predominantlyin the nucleus by immunostaining with the aforementioned antibodies (8).Given that HD1 and RbAp48 are the major proteins eluted from the K-trapmatrix (FIG. 2B), it is likely that they interact directly with oneanother.

We extended the results obtained with the endogenous protein byexpressing recombinant FLAG epitope tagged HD1 (HD1-F) in Jurkat Tcells. Anti-FLAG immunoprecipitates from cells transfected withpBJ5/HD1-F contained histone deacetylase activity that was sensitive toboth trapoxin and trichostatin (FIG. 4C). Histone deacetylase activitywas not precipitated when the antibody was blocked with excess FLAGpeptide (15). Interestingly, endogenous RbAp48 did not coprecipitatewith overexpressed HD1-F (8), demonstrating that RbAp48 is not requiredfor either histone deacetylase or trapoxin binding activity. The resultis consistent with the idea that RbAp48 serves a targeting rather thanan enzymatic function. Finally, lysates from cells transfected withpBJ5/HD1-F were incubated with the K-trap affinity matrix in thepresence or absence of trapoxin and trichostatin. Protein immunoblotanalysis demonstrated an interaction between recombinant HD1-F and theK-trap affinity matrix that was fully competed by nanomolarconcentrations of trapoxin or trichostatin (FIG. 4D).

HD1 is 60% identical to the protein encoded by the yeast RPD3 gene,which was isolated in four independent mutant suppressor screensdesigned to identify transcriptional repressors (13, 16, 17, 18, 19). Nobiochemical function for the yeast protein has previously beenpostulated. A negative regulator of the TRK2 gene, RPD3 is necessary forthe transcriptional repression of several genes whose expression isregulated according to specific environmental conditions. Loss of RPD3also leads to decreased transcriptional activation of certain genes, butthis effect may be indirect (13, 17). Although RPD3 had yet to beimplicated in silencing at telmomeres or the mating loci, the fact thatsilencing is eliminated by point mutations in specific lysine residuesnear the NH₂-terminus of histones H3 and H4 suggests that lysinedeacetylation may contribute to the maintenance of silenced chromatin(20, 21, 22, 23). Indeed, silencing at telomeres and the mating loci hasbeen correlated with the presence of hypoacetylated histones, and sirmutants which are defective in silencing show a corresponding increasein the extent of histone acetylation at these loci (24). The SIR3 andSIR4 proteins have been shown to interact with a bacterially expressedhistone H4 NH₂-terminal domain in vitro (25), and it is possible thatdeacetylation of one or more lysine residues is required for thisinteraction in vivo. Our results further support a role for histonedeacetylase as a transcriptional regulator and establish a biochemicalconnection to the genetic studies that originally characterized RPD3.

How does inhibition of histone deacetylase in mammalian cells lead toGI. and G₂ phase cell cycle arrest? One possibility is that specificcell cycle regulatory proteins such as the cyclin dependent kinaseinhibitors are transcriptionally upregulated in response to histonedeacetylase inactivation. Alternatively, cell cycle checkpoints mayexist that monitor histone acetylation or higher-order chromatinstructure. It should now be possible to study the regulation of histonedeacetylase during the cell cycle, its substrate specificity, and themechanism by which it is targeted to specific regions of the genome.

EXAMPLE 2

3H-Trapoxin was prepared from (S,S)-threitol acetonide (9) by totalsynthesis, as outlined in FIGS. 8A-8C.

As shown in FIG. 8A, (S,S)-threitol acetonide (9) was monoprotected bytreatment with triisopropylsilylchloride (TIPSCl) and sodium hydride intetrahydrofuran (THF). The free alcohol was then subjected to Swernoxidation. Wittig reaction of the resulting aldehyde gave compound 10 ingood yield for the three steps. Compound 10 was then hydrogenated withdeprotection of the primary alcohol, which was then converted to thebromide 11 in excellent yield. Bromide 11 was converted to theorganocuprate and reacted with (S)-serine β-lactone to yield thebenzyloxycarbonyl-(Cbz) protected amino acid 12.

As shown in FIG. 8B, 12 was coupled to tripeptide methyl ester 14, andthe methyl ester was saponified. The amino acid was then cyclized andthe silyl protecting group was removed to yield cyclotetrapeptide 18 in51% yield.

Cyclotetrapeptide 18 was tritiated, as shown in FIG. 8C, by oxidation ofthe primary alcohol with the Dess-Martin reagent, and the aldehyde wasreduced with tritiated sodium borohydride to provide tritiated 18, whichwas converted to [³H]Trapoxin B by tosylation of the primary alcohol,deprotection of the diol, epoxide ring closure, and oxidation of thesecondary alcohol to yield the desired compound. Non-radiolabelled 18was converted to [³H]Trapoxin B, via tosylate 19, in 68% overall yield.

EXAMPLE 3

K-Trap was prepared from (S,S)-threitol acetonide (9) by totalsynthesis, as outlined in FIGS. 9A-9C. As shown in FIG. 9A,monoprotection and Swern oxidation of 9 yielded the aldehyde as above.Wittig homologation yielded carboxylic acid 20, which was converted tothe mixed anhydride and treated with lithiated oxazolidinone 21 toprovide 22 in excellent yield. Deprotection of the primary alcohol andconversion to the tosylate were followed by treatment of the potassiumenolate with trisylazide according to the method of Evans to effectelectrophilic azide transfer in good overall yield andstereoselectivity, providing compound 23. Removal of the chiralauxiliary and catalytic reduction of the azido function, withhydrogenation of the olefin, provided amino acid 24, which wasN-protected to give the Fmoc derivative 25 in high overall yield.

Referring to FIG. 9B, protected amino acid 25 was coupled to tripeptidemethyl ester 26. The methyl ester was saponified to yield 27, which wascyclized under high-dilution conditions to provide cyclotetrapeptide 28in 58% yield.

As shown in FIG. 9C, compound 28 was converted to K-trap (29) bydeprotection of the diol, base-promoted epoxide closure, and oxidationof the secondary alcohol to provide K-trap (29) in good overall yield.The K-trap affinity matrix 30 was provided by palladium-catalyzedremoval of the allyloxycarbonyl (Alloc) group from the lysine residue of29, and immobilization on Affigel 10.

References and Notes

1. B. M. Turner, Cell 75, 5-8 (1993).

2. D. Y. Lee, J. J. Hayes, D. Pruss, A. P. Wolffe, Cell 72, 73-84 (1993)

3. S. Kelff, E. D Andrulis, C. W. Anderson, R. Stemglanz, J. Biol. Chem.270, 24674 24677(1995).

4. M. Yoshida, S. Horinouchi, T. Beppu, Bioessays 17, 423-30 (1995)

5. M. Kijima, M. Yoshida, K. Sugita, S. Horinouchi, T. Beppu, J. Biol.Chem. 268, 22429-35 (1993).

6. J. Tauton, J. L. Collins, S. L. Schreiber manuscript in preparation.

8. J. Taunton, C. A. Hassig, S. L. Schreiber, unpublished results.

11. Y. W. Qian, et al., Nature 364, 648-52 (1993).

12. E. J. Neer, C. J. Schmidt, R. Nambudripad, T. F. Smith, Nature 371,297-300 (1994).

13. M. Vidal, R. F. Gaber, Mol. Cell. Biol. 11, 6317-27 (1991). .

15. Control experiments indicated that competitor peptides had no effecton histone deacetylase activity per se.

16. K. Nasmuth, D. J. Stillman, D. Kipling, Cell 48, 579-87 (1987).

17. D. J. Stillman, S. Dorland, Y. Yu, Genetics 136, 781-8 (1994).

18. E. A. McKenzie, et al., Mol. Gen. Genet. 240, 374-86 (1993).

19. K. S. Bowdish, A. P. Mitchell, Mol. Cell. Biol. 13, 2172-81 (1993).

20. L. M. Johnson, P. S. Kayne, E. S. Kahn, M. Grunstein, Proc. Natl.Acad. Sci. U.S.A. 87, 6286-90 (1990).

21. P. C. Megee, B. A. Morgan, B. A. Mittman, M. M. Smith, Science 247,841-5 (1990).

22. E. C. Park, J. W. Szostak, Mol. Cell. Biol. 10, 49324 (1990).

23. O. M. Aparicio, B. L. Billington, D. E. Gottschling, Cell 66,1279-87 (1991).

24. M. Braunstein, A. B. Rose, S. G. Holmes, C. D. Allis, J. R. Borach,Genes Dev. 7, 592-604 (1993).

25. A. Hecht, T. Laroche, S. Strahl Bolsinger, S. M. Gasser, M.Grunstein, Cell 80, 583-92 (1995).

26. N. A. Clipstone, G. R. Crabtree, Nature 357, 695-7 (1992).

All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims.

23 1 1449 DNA Homo sapiens CDS (1)..(1446) 1 atg gcg cag acg cag ggc acccgg agg aaa gtc tgt tac tac tac gac 48 Met Ala Gln Thr Gln Gly Thr ArgArg Lys Val Cys Tyr Tyr Tyr Asp 1 5 10 15 ggg gat gtt gga aat tac tattat gga caa ggc cac cca atg aag cct 96 Gly Asp Val Gly Asn Tyr Tyr TyrGly Gln Gly His Pro Met Lys Pro 20 25 30 cac cga atc cgc atg act cat aatttg ctg ctc aac tat ggt ctc tac 144 His Arg Ile Arg Met Thr His Asn LeuLeu Leu Asn Tyr Gly Leu Tyr 35 40 45 cga aaa atg gaa atc tat cgc cct cacaaa gcc aat gct gag gag atg 192 Arg Lys Met Glu Ile Tyr Arg Pro His LysAla Asn Ala Glu Glu Met 50 55 60 acc aag tac cac agc gat gac tac att aaattc ttg cgc tcc atc cgt 240 Thr Lys Tyr His Ser Asp Asp Tyr Ile Lys PheLeu Arg Ser Ile Arg 65 70 75 80 cca gat aac atg tcg gag tac agc aag cagatg cag aga ttc aac gtt 288 Pro Asp Asn Met Ser Glu Tyr Ser Lys Gln MetGln Arg Phe Asn Val 85 90 95 ggt gag gac tgt cca gta ttc gat ggc ctg tttgag ttc tgt cag ttg 336 Gly Glu Asp Cys Pro Val Phe Asp Gly Leu Phe GluPhe Cys Gln Leu 100 105 110 tct act ggt ggt tct gtg gca agt gct gtg aaactt aat aag cag cag 384 Ser Thr Gly Gly Ser Val Ala Ser Ala Val Lys LeuAsn Lys Gln Gln 115 120 125 acg gac atc gct gtg aat tgg gct ggg ggg ctgcac cat gca aag aag 432 Thr Asp Ile Ala Val Asn Trp Ala Gly Gly Leu HisHis Ala Lys Lys 130 135 140 tcc gag gca tct ggc ttc tgt tac gtc aat gatatc gtc ttg gcc atc 480 Ser Glu Ala Ser Gly Phe Cys Tyr Val Asn Asp IleVal Leu Ala Ile 145 150 155 160 ctg gaa ctg cta aag tat cac cag agg gtgctg tac att gac att gat 528 Leu Glu Leu Leu Lys Tyr His Gln Arg Val LeuTyr Ile Asp Ile Asp 165 170 175 att cac cat ggt gac ggc gtg gaa gag gccttc tac acc acg gac cgg 576 Ile His His Gly Asp Gly Val Glu Glu Ala PheTyr Thr Thr Asp Arg 180 185 190 gtc atg act gtg tcc ttt cat aag tat ggagag tac ttc cca gga act 624 Val Met Thr Val Ser Phe His Lys Tyr Gly GluTyr Phe Pro Gly Thr 195 200 205 ggg gac cta cgg gat atc ggg gct ggc aaaggc aag tat tat gct gtt 672 Gly Asp Leu Arg Asp Ile Gly Ala Gly Lys GlyLys Tyr Tyr Ala Val 210 215 220 aac tac ccg ctc cga gac ggg att gat gacgag tcc tat gag gcc att 720 Asn Tyr Pro Leu Arg Asp Gly Ile Asp Asp GluSer Tyr Glu Ala Ile 225 230 235 240 ttc aag ccg gtc atg tcc aaa gta atggag atg ttc cag cct agt gcg 768 Phe Lys Pro Val Met Ser Lys Val Met GluMet Phe Gln Pro Ser Ala 245 250 255 gtg gtc tta cag tgt ggc tca gac tcccta tct ggg gat cgg tta ggt 816 Val Val Leu Gln Cys Gly Ser Asp Ser LeuSer Gly Asp Arg Leu Gly 260 265 270 tgc ttc aat cta act atc aaa gga cacgcc aag tgt gtg gaa ttt gtc 864 Cys Phe Asn Leu Thr Ile Lys Gly His AlaLys Cys Val Glu Phe Val 275 280 285 aag agc ttt aac ctg cct atg ctg atgctg gga ggc ggt ggt tac acc 912 Lys Ser Phe Asn Leu Pro Met Leu Met LeuGly Gly Gly Gly Tyr Thr 290 295 300 att cgt aac gtt gcc cgg tgc tgg acatat gag aca gct gtg gcc ctg 960 Ile Arg Asn Val Ala Arg Cys Trp Thr TyrGlu Thr Ala Val Ala Leu 305 310 315 320 gat acg gag atc cct aat gag cttcca tac aat gac tac ttt gaa tac 1008 Asp Thr Glu Ile Pro Asn Glu Leu ProTyr Asn Asp Tyr Phe Glu Tyr 325 330 335 ttt gga cca gat ttc aag ctc cacatc agt cct tcc aat atg act aac 1056 Phe Gly Pro Asp Phe Lys Leu His IleSer Pro Ser Asn Met Thr Asn 340 345 350 cag aac acg aat gag tac ctg gagaag atc aaa cag cga ctg ttt gag 1104 Gln Asn Thr Asn Glu Tyr Leu Glu LysIle Lys Gln Arg Leu Phe Glu 355 360 365 aac ctt aga atg ctg ccg cac gcacct ggg gtc caa atg cag gcg att 1152 Asn Leu Arg Met Leu Pro His Ala ProGly Val Gln Met Gln Ala Ile 370 375 380 cct gag gac gcc atc cct gag gagagt ggc gat gag gac gaa gac gac 1200 Pro Glu Asp Ala Ile Pro Glu Glu SerGly Asp Glu Asp Glu Asp Asp 385 390 395 400 cct gac aag cgc atc tcg atctgc tcc tct gac aaa cga att gcc tgt 1248 Pro Asp Lys Arg Ile Ser Ile CysSer Ser Asp Lys Arg Ile Ala Cys 405 410 415 gag gaa gag ttc tcc gat tctgaa gag gag gga gag ggg ggc cgc aag 1296 Glu Glu Glu Phe Ser Asp Ser GluGlu Glu Gly Glu Gly Gly Arg Lys 420 425 430 aac tct tcc aac ttc aaa aaagcc aag aga gtc aaa aca gag gat gaa 1344 Asn Ser Ser Asn Phe Lys Lys AlaLys Arg Val Lys Thr Glu Asp Glu 435 440 445 aaa gag aaa gac cca gag gagaag aaa gaa gtc acc gaa gag gag aaa 1392 Lys Glu Lys Asp Pro Glu Glu LysLys Glu Val Thr Glu Glu Glu Lys 450 455 460 acc aag gag gag aag cca gaagcc aaa ggg gtc aag gag gag gtc aag 1440 Thr Lys Glu Glu Lys Pro Glu AlaLys Gly Val Lys Glu Glu Val Lys 465 470 475 480 ttg gcc tga 1449 Leu Ala2 379 DNA Homo sapiens 2 attgacttcc tgcagagagt cagccccacc aatatgcaaggcttcaccaa gagtcttaat 60 gccttcaacg taggcgatga ctgcccagtg tttcccgggctctttgagtt ctgctcgcgt 120 tacacaggcg catctctgca aggagcaacc cagctgaacaacaagatctg tgatattgcc 180 attaactggg ctggtggtct gcaccatgcc tagaagtttgaggcctctgg cttctgctat 240 gtcaacgaca ttgtgtttgg catcctggag ctgctcaagtaccaccctcg ggtgctctac 300 attgacattg acatccacca tggtgacggg gttcaagaagctttctacct cactgaccgg 360 gtcatgacgg tgtcctttc 379 3 375 DNA Homosapiens 3 tactactgtc tgaacgtgcc cctgcggatg ggcattgatg accagagttacaagcacctt 60 ttccagccgg ttatcaacca ggtagtggac ttctaccaac ccacgtgcattgtgctccag 120 tgtggagctg actctctggg ctgtgatcga ttgggctgct ttaacctcagcatccgaggg 180 catggggaat gcgttgaata tgtcaagagc ttcaatatcc ctctactcgtgctgggtggt 240 ggtggttata ctgtccgaaa tgttgcccgc tgctggacat atgagacatcgctgctggta 300 gaagaggcca ttagtgagga gcttccctat agtgaatact tcgagtactttgccccagac 360 ttcacacttc atcca 375 4 227 DNA Homo sapiens 4 ggtcatgctaaatgtgtaga agttgtaaaa acttttaact taccattact gatgcttgga 60 ggaggtggctacacaatccg taatgttgct cgatgttgga catatgagac tgcagttgcc 120 cttgattgtgagattcccaa tgagttgcca tataatgatt actttgagta ttttggacca 180 gacttcaaactgcatattag tccttcaaac atgacaaacc agaacac 227 5 482 PRT Homo sapiens 5Met Ala Gln Thr Gln Gly Thr Arg Arg Lys Val Cys Tyr Tyr Tyr Asp 1 5 1015 Gly Asp Val Gly Asn Tyr Tyr Tyr Gly Gln Gly His Pro Met Lys Pro 20 2530 His Arg Ile Arg Met Thr His Asn Leu Leu Leu Asn Tyr Gly Leu Tyr 35 4045 Arg Lys Met Glu Ile Tyr Arg Pro His Lys Ala Asn Ala Glu Glu Met 50 5560 Thr Lys Tyr His Ser Asp Asp Tyr Ile Lys Phe Leu Arg Ser Ile Arg 65 7075 80 Pro Asp Asn Met Ser Glu Tyr Ser Lys Gln Met Gln Arg Phe Asn Val 8590 95 Gly Glu Asp Cys Pro Val Phe Asp Gly Leu Phe Glu Phe Cys Gln Leu100 105 110 Ser Thr Gly Gly Ser Val Ala Ser Ala Val Lys Leu Asn Lys GlnGln 115 120 125 Thr Asp Ile Ala Val Asn Trp Ala Gly Gly Leu His His AlaLys Lys 130 135 140 Ser Glu Ala Ser Gly Phe Cys Tyr Val Asn Asp Ile ValLeu Ala Ile 145 150 155 160 Leu Glu Leu Leu Lys Tyr His Gln Arg Val LeuTyr Ile Asp Ile Asp 165 170 175 Ile His His Gly Asp Gly Val Glu Glu AlaPhe Tyr Thr Thr Asp Arg 180 185 190 Val Met Thr Val Ser Phe His Lys TyrGly Glu Tyr Phe Pro Gly Thr 195 200 205 Gly Asp Leu Arg Asp Ile Gly AlaGly Lys Gly Lys Tyr Tyr Ala Val 210 215 220 Asn Tyr Pro Leu Arg Asp GlyIle Asp Asp Glu Ser Tyr Glu Ala Ile 225 230 235 240 Phe Lys Pro Val MetSer Lys Val Met Glu Met Phe Gln Pro Ser Ala 245 250 255 Val Val Leu GlnCys Gly Ser Asp Ser Leu Ser Gly Asp Arg Leu Gly 260 265 270 Cys Phe AsnLeu Thr Ile Lys Gly His Ala Lys Cys Val Glu Phe Val 275 280 285 Lys SerPhe Asn Leu Pro Met Leu Met Leu Gly Gly Gly Gly Tyr Thr 290 295 300 IleArg Asn Val Ala Arg Cys Trp Thr Tyr Glu Thr Ala Val Ala Leu 305 310 315320 Asp Thr Glu Ile Pro Asn Glu Leu Pro Tyr Asn Asp Tyr Phe Glu Tyr 325330 335 Phe Gly Pro Asp Phe Lys Leu His Ile Ser Pro Ser Asn Met Thr Asn340 345 350 Gln Asn Thr Asn Glu Tyr Leu Glu Lys Ile Lys Gln Arg Leu PheGlu 355 360 365 Asn Leu Arg Met Leu Pro His Ala Pro Gly Val Gln Met GlnAla Ile 370 375 380 Pro Glu Asp Ala Ile Pro Glu Glu Ser Gly Asp Glu AspGlu Asp Asp 385 390 395 400 Pro Asp Lys Arg Ile Ser Ile Cys Ser Ser AspLys Arg Ile Ala Cys 405 410 415 Glu Glu Glu Phe Ser Asp Ser Glu Glu GluGly Glu Gly Gly Arg Lys 420 425 430 Asn Ser Ser Asn Phe Lys Lys Ala LysArg Val Lys Thr Glu Asp Glu 435 440 445 Lys Glu Lys Asp Pro Glu Glu LysLys Glu Val Thr Glu Glu Glu Lys 450 455 460 Thr Lys Glu Glu Lys Pro GluAla Lys Gly Val Lys Glu Glu Val Lys 465 470 475 480 Leu Ala 6 133 PRTHomo sapiens 6 Ile Asp Phe Leu Gln Arg Val Ser Pro Thr Asn Met Gln GlyPhe Thr 1 5 10 15 Lys Ser Leu Asn Ala Phe Asn Val Gly Asp Asp Cys ProVal Phe Pro 20 25 30 Gly Leu Phe Glu Phe Cys Ser Arg Tyr Thr Gly Ala SerLeu Gln Gly 35 40 45 Ala Thr Gln Leu Asn Asn Lys Ile Cys Asp Ile Ala IleAsn Trp Ala 50 55 60 Gly Gly Leu His His Ala Lys Lys Phe Glu Ala Ser GlyPhe Cys Tyr 65 70 75 80 Val Asn Asp Ile Val Phe Gly Ile Leu Glu Leu LeuLys Tyr His Pro 85 90 95 Arg Val Leu Tyr Ile Asp Ile Asp Ile His His GlyAsp Gly Val Gln 100 105 110 Glu Ala Phe Tyr Leu Thr Asp Arg Val Met ThrVal Ser Phe Pro Gln 115 120 125 Ile Arg Glu Ile Tyr 130 7 125 PRT Homosapiens 7 Tyr Tyr Cys Leu Asn Val Pro Leu Arg Met Gly Ile Asp Asp GlnSer 1 5 10 15 Tyr Lys His Leu Phe Gln Pro Val Ile Asn Gln Val Val AspPhe Tyr 20 25 30 Gln Pro Thr Cys Ile Val Leu Gln Cys Gly Ala Asp Ser LeuGly Cys 35 40 45 Asp Arg Leu Gly Cys Phe Asn Leu Ser Ile Arg Gly His GlyGlu Cys 50 55 60 Val Glu Tyr Val Lys Ser Phe Asn Ile Pro Leu Leu Val LeuGly Gly 65 70 75 80 Gly Gly Tyr Thr Val Arg Asn Val Ala Arg Cys Trp ThrTyr Glu Thr 85 90 95 Ser Leu Leu Val Glu Glu Ala Ile Ser Glu Glu Leu ProTyr Ser Glu 100 105 110 Tyr Phe Glu Tyr Phe Ala Pro Asp Phe Thr Leu HisPro 115 120 125 8 80 PRT Homo sapiens 8 Asn Leu Leu Val Leu Gly His AlaLys Cys Val Glu Val Val Lys Thr 1 5 10 15 Phe Asn Leu Pro Leu Leu MetLeu Gly Gly Gly Gly Tyr Thr Ile Arg 20 25 30 Asn Val Ala Arg Cys Trp ThrTyr Glu Thr Ala Val Ala Leu Asp Cys 35 40 45 Glu Ile Pro Asn Glu Leu ProTyr Asn Asp Tyr Phe Glu Tyr Phe Gly 50 55 60 Pro Asp Phe Lys Leu His IleSer Pro Ser Asn Met Thr Asn Gln Asn 65 70 75 80 9 433 PRT Saccharomycescerevisiae 9 Met Val Tyr Glu Ala Thr Pro Phe Asp Pro Ile Thr Val Lys ProSer 1 5 10 15 Asp Lys Arg Arg Val Ala Tyr Phe Tyr Asp Ala Asp Val GlyAsn Tyr 20 25 30 Ala Tyr Gly Ala Gly His Pro Met Lys Pro His Arg Ile ArgMet Ala 35 40 45 His Ser Leu Ile Met Asn Tyr Gly Leu Tyr Lys Lys Met GluIle Tyr 50 55 60 Arg Ala Lys Pro Ala Thr Lys Gln Glu Met Cys Gln Phe HisThr Asp 65 70 75 80 Glu Tyr Ile Asp Phe Leu Ser Arg Val Thr Pro Asp AsnLeu Glu Met 85 90 95 Phe Lys Arg Glu Ser Val Lys Phe Asn Val Gly Asp AspCys Pro Val 100 105 110 Phe Asp Gly Leu Tyr Glu Tyr Cys Ser Ile Ser GlyGly Gly Ser Met 115 120 125 Glu Gly Ala Ala Arg Leu Asn Arg Gly Lys CysAsp Val Ala Val Asn 130 135 140 Tyr Ala Gly Gly Leu His His Ala Lys LysSer Glu Ala Ser Gly Phe 145 150 155 160 Cys Tyr Leu Asn Asp Ile Val LeuGly Ile Ile Glu Leu Leu Arg Tyr 165 170 175 His Pro Arg Val Leu Tyr IleAsp Ile Asp Val His His Gly Asp Gly 180 185 190 Val Glu Glu Ala Phe TyrThr Thr Asp Arg Val Met Thr Cys Ser Phe 195 200 205 His Lys Tyr Gly GluPhe Phe Pro Gly Thr Gly Glu Leu Arg Asp Ile 210 215 220 Gly Val Gly AlaGly Lys Asn Tyr Ala Val Asn Val Pro Leu Arg Asp 225 230 235 240 Gly IleAsp Asp Ala Thr Tyr Arg Ser Val Phe Glu Pro Val Ile Lys 245 250 255 LysIle Met Glu Trp Tyr Gln Pro Ser Ala Val Val Leu Gln Cys Gly 260 265 270Gly Asp Ser Leu Ser Gly Asp Arg Leu Gly Cys Phe Asn Leu Ser Met 275 280285 Glu Gly His Ala Asn Cys Val Asn Tyr Val Lys Ser Phe Gly Ile Pro 290295 300 Met Met Val Val Gly Gly Gly Gly Tyr Thr Met Arg Asn Val Ala Arg305 310 315 320 Thr Trp Cys Phe Glu Thr Gly Leu Leu Asn Asn Val Val LeuAsp Lys 325 330 335 Asp Leu Pro Tyr Asn Glu Tyr Tyr Glu Tyr Tyr Gly ProAsp Tyr Lys 340 345 350 Leu Ser Val Arg Pro Ser Asn Met Phe Asn Val AsnThr Pro Glu Tyr 355 360 365 Leu Asp Lys Val Met Thr Asn Ile Phe Ala AsnLeu Glu Asn Thr Lys 370 375 380 Tyr Ala Pro Ser Val Gln Leu Asn His ThrPro Arg Asp Ala Glu Asp 385 390 395 400 Leu Gly Asp Val Glu Glu Asp SerAla Glu Ala Lys Asp Thr Lys Gly 405 410 415 Gly Ser Gly Tyr Ala Arg AspLeu His Val Glu His Asp Asn Glu Phe 420 425 430 Tyr 10 480 PRT Xenopuslaevis 10 Met Ala Leu Thr Leu Gly Thr Lys Lys Lys Val Cys Tyr Tyr TyrAsp 1 5 10 15 Gly Asp Val Gly Asn Tyr Tyr Tyr Gly Gln Gly His Pro MetLys Pro 20 25 30 His Arg Ile Arg Met Thr His Asn Leu Leu Leu Asn Tyr GlyLeu Tyr 35 40 45 Arg Lys Met Glu Ile Phe Arg Pro His Lys Ala Ser Ala GluAsp Met 50 55 60 Thr Lys Tyr His Ser Asp Asp Tyr Ile Lys Phe Leu Arg SerIle Arg 65 70 75 80 Pro Asp Asn Met Ser Glu Tyr Ser Lys Gln Met Gln ArgPhe Asn Val 85 90 95 Gly Glu Asp Cys Pro Val Phe Asp Gly Leu Phe Glu PheCys Gln Leu 100 105 110 Ser Ala Gly Gly Ser Val Ala Ser Ala Val Lys LeuAsn Lys Gln Gln 115 120 125 Thr Asp Ile Ser Val Asn Trp Ser Gly Gly LeuHis His Ala Lys Lys 130 135 140 Ser Glu Ala Ser Gly Phe Cys Tyr Val AsnAsp Ile Val Leu Ala Ile 145 150 155 160 Leu Glu Leu Leu Lys Tyr His GlnArg Val Val Tyr Ile Asp Ile Asp 165 170 175 Ile His His Gly Asp Gly ValGlu Glu Ala Phe Tyr Thr Thr Asp Arg 180 185 190 Val Met Thr Val Ser PheHis Lys Tyr Gly Glu Tyr Phe Pro Gly Thr 195 200 205 Gly Asp Leu Arg AspIle Gly Ala Gly Lys Gly Lys Tyr Tyr Ala Val 210 215 220 Asn Tyr Ala LeuArg Asp Gly Ile Asp Asp Glu Ser Tyr Glu Ala Ile 225 230 235 240 Phe LysPro Val Met Ser Lys Val Met Glu Met Phe Gln Pro Ser Ala 245 250 255 ValVal Leu Gln Cys Gly Ala Asp Ser Leu Ser Gly Asp Arg Leu Gly 260 265 270Cys Phe Asn Leu Thr Ile Lys Gly His Ala Lys Cys Val Glu Phe Ile 275 280285 Lys Thr Phe Asn Leu Pro Leu Leu Met Leu Gly Gly Gly Gly Tyr Thr 290295 300 Ile Arg Asn Val Ala Arg Cys Trp Thr Tyr Glu Thr Ala Val Ala Leu305 310 315 320 Asp Ser Glu Ile Pro Asn Glu Leu Pro Tyr Asn Asp Tyr PheGlu Tyr 325 330 335 Phe Gly Pro Asp Phe Lys Leu His Ile Ser Pro Ser AsnMet Thr Asn 340 345 350 Gln Asn Thr Asn Glu Tyr Leu Glu Lys Ile Lys GlnArg Leu Phe Glu 355 360 365 Asn Leu Arg Met Leu Pro His Ala Pro Gly ValGln Met Gln Ala Val 370 375 380 Ala Glu Asp Ser Ile His Asp Asp Ser GlyGlu Glu Asp Glu Asp Asp 385 390 395 400 Pro Asp Lys Arg Ile Ser Ile ArgSer Ser Asp Lys Arg Ile Ala Cys 405 410 415 Asp Glu Glu Phe Ser Asp SerGlu Asp Glu Gly Glu Gly Gly Arg Lys 420 425 430 Asn Val Ala Asn Phe LysLys Val Lys Arg Val Lys Thr Glu Glu Glu 435 440 445 Lys Glu Gly Glu AspLys Lys Asp Val Lys Glu Glu Glu Lys Ala Lys 450 455 460 Asp Glu Lys ThrAsp Ser Lys Arg Val Lys Glu Glu Thr Lys Ser Val 465 470 475 480 11 1278DNA Homo sapiens CDS (1)..(1278) 11 atg gcc gac aag gaa gca gcc ttc gacgac gca gtg gaa gaa cga gtg 48 Met Ala Asp Lys Glu Ala Ala Phe Asp AspAla Val Glu Glu Arg Val 1 5 10 15 atc aac gag gaa tac aaa ata tgg aaaaag aac acc cct ttt ctt tat 96 Ile Asn Glu Glu Tyr Lys Ile Trp Lys LysAsn Thr Pro Phe Leu Tyr 20 25 30 gat ttg gtg atg acc cat gct ctg gag tggccc agc cta act gcc cag 144 Asp Leu Val Met Thr His Ala Leu Glu Trp ProSer Leu Thr Ala Gln 35 40 45 tgg ctt cca gat gta acc aga cca gaa ggg aaagat ttc agc att cat 192 Trp Leu Pro Asp Val Thr Arg Pro Glu Gly Lys AspPhe Ser Ile His 50 55 60 cga ctt gtc ctg ggg aca cac aca tcg gat gaa caaaac cat ctt gtt 240 Arg Leu Val Leu Gly Thr His Thr Ser Asp Glu Gln AsnHis Leu Val 65 70 75 80 ata gcc agt gtg cag ctc cct aat gat gat gct cagttt gat gcg tca 288 Ile Ala Ser Val Gln Leu Pro Asn Asp Asp Ala Gln PheAsp Ala Ser 85 90 95 cac tac gac agt gag aaa gga gaa ttt gga ggt ttt ggttca gtt agt 336 His Tyr Asp Ser Glu Lys Gly Glu Phe Gly Gly Phe Gly SerVal Ser 100 105 110 gga aaa att gaa ata gaa atc aag atc aac cat gaa ggagaa gta aac 384 Gly Lys Ile Glu Ile Glu Ile Lys Ile Asn His Glu Gly GluVal Asn 115 120 125 agg gcc cgt tat atg ccc cag aac cct tgt atc atc gcaaca aag act 432 Arg Ala Arg Tyr Met Pro Gln Asn Pro Cys Ile Ile Ala ThrLys Thr 130 135 140 cct tcc agt gat gtt ctt gtc ttt gac tat aca aaa catcct tct aaa 480 Pro Ser Ser Asp Val Leu Val Phe Asp Tyr Thr Lys His ProSer Lys 145 150 155 160 cca gat cct tct gga gag tgc aac cca gac ttg cgtctc cgt gga cat 528 Pro Asp Pro Ser Gly Glu Cys Asn Pro Asp Leu Arg LeuArg Gly His 165 170 175 cag aag gaa ggc tat ggg ctt tct tgg aac cca aatctc agt ggg cac 576 Gln Lys Glu Gly Tyr Gly Leu Ser Trp Asn Pro Asn LeuSer Gly His 180 185 190 tta ctt agt gct tca gat gac cat acc atc tgc ctgtgg gac atc agt 624 Leu Leu Ser Ala Ser Asp Asp His Thr Ile Cys Leu TrpAsp Ile Ser 195 200 205 gcc gtt cca aag gag gga aaa gtg gta gat gcg aagacc atc ttt aca 672 Ala Val Pro Lys Glu Gly Lys Val Val Asp Ala Lys ThrIle Phe Thr 210 215 220 ggg cat acg gca gta gta gaa gat gtt tcc tgg catcta ctc cat gag 720 Gly His Thr Ala Val Val Glu Asp Val Ser Trp His LeuLeu His Glu 225 230 235 240 tct ctg ttt ggg tca gtt gct gat gat cag aaactt atg att tgg gat 768 Ser Leu Phe Gly Ser Val Ala Asp Asp Gln Lys LeuMet Ile Trp Asp 245 250 255 act cgt tca aac aat act tcc aaa cca agc cactca gtt gat gct cac 816 Thr Arg Ser Asn Asn Thr Ser Lys Pro Ser His SerVal Asp Ala His 260 265 270 act gct gaa gtg aac tgc ctt tct ttc aat ccttat agt gag ttc att 864 Thr Ala Glu Val Asn Cys Leu Ser Phe Asn Pro TyrSer Glu Phe Ile 275 280 285 ctt gcc aca gga tca gct gac aag act gtt gccttg tgg gat ctg aga 912 Leu Ala Thr Gly Ser Ala Asp Lys Thr Val Ala LeuTrp Asp Leu Arg 290 295 300 aat ctg aaa ctt aag ttg cat tcc ttt gag tcacat aag gat gaa ata 960 Asn Leu Lys Leu Lys Leu His Ser Phe Glu Ser HisLys Asp Glu Ile 305 310 315 320 ttc cag gtt cag tgg tca cct cac aat gagact att tta gct tcc agt 1008 Phe Gln Val Gln Trp Ser Pro His Asn Glu ThrIle Leu Ala Ser Ser 325 330 335 ggt act gat cgc aga ctg aat gtc tgg gattta agt aaa att gga gag 1056 Gly Thr Asp Arg Arg Leu Asn Val Trp Asp LeuSer Lys Ile Gly Glu 340 345 350 gaa caa tcc cca gaa gat gca gaa gac gggcca cca gag ttg ttg ttt 1104 Glu Gln Ser Pro Glu Asp Ala Glu Asp Gly ProPro Glu Leu Leu Phe 355 360 365 att cat ggt ggt cat act gcc aag ata tctgat ttc tcc tgg aat ccc 1152 Ile His Gly Gly His Thr Ala Lys Ile Ser AspPhe Ser Trp Asn Pro 370 375 380 aat gaa cct tgg gtg att tgt tct gta tcagaa gac aat atc atg caa 1200 Asn Glu Pro Trp Val Ile Cys Ser Val Ser GluAsp Asn Ile Met Gln 385 390 395 400 gtg tgg caa atg gca gag aac att tataat gat gaa gac cct gaa gga 1248 Val Trp Gln Met Ala Glu Asn Ile Tyr AsnAsp Glu Asp Pro Glu Gly 405 410 415 agc gtg gat cca gaa gga caa ggg tcctag 1278 Ser Val Asp Pro Glu Gly Gln Gly Ser 420 425 12 69 PRTArtificial Sequence Description of Artificial Sequence Synthetic motifconsensus sequence 12 Asp Xaa Xaa Xaa Asn Xaa Xaa Gly Gly Leu His HisAla Lys Lys Xaa 1 5 10 15 Glu Ala Ser Gly Phe Cys Tyr Xaa Asn Asp IleVal Xaa Xaa Ile Xaa 20 25 30 Glu Leu Leu Xaa Tyr His Xaa Arg Val Xaa TyrIle Asp Xaa Asp Xaa 35 40 45 His His Gly Asp Gly Xaa Glu Glu Ala Phe TyrXaa Thr Asp Arg Val 50 55 60 Met Thr Xaa Ser Phe 65 13 68 PRT ArtificialSequence Description of Artificial Sequence Synthetic motif consensussequence 13 Asp Ile Ala Xaa Asn Trp Ala Gly Gly Leu His His Ala Lys LysXaa 1 5 10 15 Glu Ala Ser Gly Phe Cys Tyr Val Asn Asp Ile Val Xaa XaaIle Leu 20 25 30 Glu Leu Leu Lys Tyr His Xaa Arg Val Leu Tyr Ile Asp IleAsp Ile 35 40 45 His His Gly Asp Gly Xaa Glu Ala Phe Tyr Xaa Thr Asp ArgVal Met 50 55 60 Thr Val Ser Phe 65 14 33 PRT Artificial SequenceDescription of Artificial Sequence Synthetic motif consensus sequence 14Cys Val Xaa Xaa Xaa Lys Xaa Phe Xaa Xaa Pro Xaa Xaa Xaa Xaa Gly 1 5 1015 Gly Gly Gly Tyr Thr Xaa Arg Asn Val Ala Arg Xaa Trp Xaa Xaa Glu 20 2530 Thr 15 33 PRT Artificial Sequence Description of Artificial SequenceSynthetic motif consensus sequence 15 Cys Val Glu Xaa Val Lys Xaa PheAsn Xaa Pro Leu Leu Xaa Leu Gly 1 5 10 15 Gly Gly Gly Tyr Thr Xaa ArgAsn Val Ala Arg Cys Trp Thr Tyr Glu 20 25 30 Thr 16 33 PRT ArtificialSequence Description of Artificial Sequence Synthetic motif consensussequence 16 Cys Val Glu Xaa Val Lys Xaa Phe Asn Xaa Pro Xaa Leu Xaa LeuGly 1 5 10 15 Gly Gly Gly Tyr Thr Xaa Arg Asn Val Ala Arg Cys Trp ThrTyr Glu 20 25 30 Thr 17 425 PRT Homo sapiens 17 Met Ala Asp Lys Glu AlaAla Phe Asp Asp Ala Val Glu Glu Arg Val 1 5 10 15 Ile Asn Glu Glu TyrLys Ile Trp Lys Lys Asn Thr Pro Phe Leu Tyr 20 25 30 Asp Leu Val Met ThrHis Ala Leu Glu Trp Pro Ser Leu Thr Ala Gln 35 40 45 Trp Leu Pro Asp ValThr Arg Pro Glu Gly Lys Asp Phe Ser Ile His 50 55 60 Arg Leu Val Leu GlyThr His Thr Ser Asp Glu Gln Asn His Leu Val 65 70 75 80 Ile Ala Ser ValGln Leu Pro Asn Asp Asp Ala Gln Phe Asp Ala Ser 85 90 95 His Tyr Asp SerGlu Lys Gly Glu Phe Gly Gly Phe Gly Ser Val Ser 100 105 110 Gly Lys IleGlu Ile Glu Ile Lys Ile Asn His Glu Gly Glu Val Asn 115 120 125 Arg AlaArg Tyr Met Pro Gln Asn Pro Cys Ile Ile Ala Thr Lys Thr 130 135 140 ProSer Ser Asp Val Leu Val Phe Asp Tyr Thr Lys His Pro Ser Lys 145 150 155160 Pro Asp Pro Ser Gly Glu Cys Asn Pro Asp Leu Arg Leu Arg Gly His 165170 175 Gln Lys Glu Gly Tyr Gly Leu Ser Trp Asn Pro Asn Leu Ser Gly His180 185 190 Leu Leu Ser Ala Ser Asp Asp His Thr Ile Cys Leu Trp Asp IleSer 195 200 205 Ala Val Pro Lys Glu Gly Lys Val Val Asp Ala Lys Thr IlePhe Thr 210 215 220 Gly His Thr Ala Val Val Glu Asp Val Ser Trp His LeuLeu His Glu 225 230 235 240 Ser Leu Phe Gly Ser Val Ala Asp Asp Gln LysLeu Met Ile Trp Asp 245 250 255 Thr Arg Ser Asn Asn Thr Ser Lys Pro SerHis Ser Val Asp Ala His 260 265 270 Thr Ala Glu Val Asn Cys Leu Ser PheAsn Pro Tyr Ser Glu Phe Ile 275 280 285 Leu Ala Thr Gly Ser Ala Asp LysThr Val Ala Leu Trp Asp Leu Arg 290 295 300 Asn Leu Lys Leu Lys Leu HisSer Phe Glu Ser His Lys Asp Glu Ile 305 310 315 320 Phe Gln Val Gln TrpSer Pro His Asn Glu Thr Ile Leu Ala Ser Ser 325 330 335 Gly Thr Asp ArgArg Leu Asn Val Trp Asp Leu Ser Lys Ile Gly Glu 340 345 350 Glu Gln SerPro Glu Asp Ala Glu Asp Gly Pro Pro Glu Leu Leu Phe 355 360 365 Ile HisGly Gly His Thr Ala Lys Ile Ser Asp Phe Ser Trp Asn Pro 370 375 380 AsnGlu Pro Trp Val Ile Cys Ser Val Ser Glu Asp Asn Ile Met Gln 385 390 395400 Val Trp Gln Met Ala Glu Asn Ile Tyr Asn Asp Glu Asp Pro Glu Gly 405410 415 Ser Val Asp Pro Glu Gly Gln Gly Ser 420 425 18 383 DNA UnknownOrganism Description of Unknown Organism R18769 nucleotide sequence 18cgatgactgc ccagtgtttc ccgggctctt tgagttctgc tcgcgttaca caggcgcatc 60tctgcaagga gcaacccagc tgaacaacaa gatctgtgat attgccatta acttggctgg 120tggcttnaac natgccanga ngtttnaggc ctctggnttc tgctatgtca acgacattgt 180gattggcatc ctggagctgc tcaagtacca ccctcgggtg ctctacattg acattgacat 240ccaccatggt gacggggttc aagaagcttt ctacctcact gaccgggtca tgacggtgtc 300ctttccacaa atacgggaaa tttacttntt ccnggggcac aggtgacatg ttntggaagt 360tcggggggca ggagagttgg ccc 383 19 213 DNA Unknown Organism Description ofUnknown Organism D31480 nucleotide sequence 19 atggcgcaga cgcagggcacccggaggaaa gtntgttact actacgacgg ggatgttgga 60 aattactatt atggacaaggccacccaatg aagcctcacc gaatccgcat gactcataat 120 ttgctgctca actatggtctctaccgaaaa atggaaatct atcgncctca caaagccaat 180 nctgaggaga tgaccaagtancacagcgat gac 213 20 313 DNA Unknown Organism Description of UnknownOrganism R98879 nucleotide sequence 20 tcctgcagag agtcagcccc accaatatgcaaggcttcac caagagtctt aatgccttca 60 acgtaggcga tgactgccca gtgtttcccgggctctttga gttctgctcg cgttacacag 120 gcgcatctct gcaaggagca acccagctgaacaacaagat ctgtgatatt gccattaact 180 gggctggtng tctgcaccat gccaagaagtttgaggcctc tggtttctgc tatgtcaacg 240 acattgtgat tggcatcctg gagctgctcaagtaccaccc tcgggtgctc tacattgaca 300 ttgacatcca cca 313 21 370 DNAUnknown Organism Description of Unknown Organism N59055 nucleotidesequence 21 ccctatagtg agtcgtattn ntnaaaacat gactcactng gntnnntacgattgggctgc 60 tttaacctca gcatccgagg gcatgggnaa tgcgttgaat atgtcaagagcttcaatatc 120 cctctactcg tgctgggtgg tggtggttat actgtccgaa atgtngcccgctgctggaca 180 tatgagacan cgctgctggt agaagaggcc attagtgagg agcttccctaatagtgaata 240 cttcgntact ttgccccaga cttcacactt catccanatg tcagcacccgcatcgagaat 300 ccagaactca cgccagtatc nggaccaaga tccgccagac aatctttgnaaacctgaagg 360 ttcttnaacc 370 22 177 DNA Unknown Organism Description ofUnknown Organism F06693 nucleotide sequence 22 aggtnatgct aaatgtgtagaagttgtaaa aacttttaac ttaccattac tgatgcttgg 60 aggaggtggc tacacaatccgtaatgttgc tcgatgttgg acatatgaga ctgcagttgc 120 ccttgattgt gagattcccaatggtaagtg ttctcattac aatatcttta ttgtatg 177 23 208 DNA Unknown OrganismDescription of Unknown Organism H05234 nucleotide sequence 23 ctacaccacggaccgggtca tgactgtgtc ctttcataag tatggagagt acttcccagg 60 gacttgggacctacgggata tcggggctgg caaaggcaag tattatgctg ttaactaccc 120 gctccgagacgggattnatg acgagtccta tgaggccatt ttcaagccgg tcatgtccaa 180 agtaatngagatgttccagc ctagtgcg 208

What is claimed is:
 1. An isolated nucleic acid encoding a histonedeacetylase gene family (HDx) polypeptide having a histone deacetylaseactivity, which HDx polypeptide comprises a ν motif represented in thegeneral formulaDIAX₁NWAGGLHHAKKX₂EASGFCYVNDIVX₃X₄ILELLKYHX₅RVLYLDIDIHHGDGX₆EAFYX₇TDRVMTVSF (SEQ ID NO: 13) and a χ motif represented inCVEX₁VKX₂FNX₃P-X₄LX₅LGGGGYTX₆RNVARCWTYET (SEQ ID NO: 16), and whichnucleic acid hybridizes under stringent conditions of 6.0×SSC at 65° C.to the nucleotide sequence designated in SEQ ID NO:
 1. 2. An isolatednucleic acid encoding an HDx polypeptide sequence or fragment thereof,which can deacetylate an acetylated histone substrate, and whichpolypeptide comprises the polypeptide sequence designated in SEQ ID NO:5.
 3. The nucleic acid of claim 1, which HDx polypeptide has a molecularweight in the range of 45-70 Kd.
 4. The nucleic acid of claim 1, whichHDx polypeptide is a fusion protein.
 5. The nucleic acid of claim 4,wherein said fusion protein includes, as a second polypeptide sequence,a polypeptide which functions as a detectable label for detecting thepresence of said fusion protein or as a matrix-binding domain forimmobilizing said fusion protein.
 6. The nucleic acid of claim 1,further comprising a transcriptional regulatory sequence operably linkedto said nucleotide sequence so as to render said nucleic acid suitablefor use as an expression vector.
 7. An expression vector, capable ofreplicating in at least one of a prokaryotic cell and eukaryotic cell,comprising the nucleic acid of claim
 6. 8. A host cell transfected withthe expression vector of claim 7 and expressing said recombinantpolypeptide.
 9. A method of producing a recombinant HDx polypeptidecomprising culturing the cell of claim 8 in a cell culture medium toexpress said recombinant polypeptide and isolating said recombinantpolypeptide from said cell culture.
 10. A recombinant transfectionsystem, comprising: (i) a gene construct including the nucleic acid ofclaim 1 and operably linked to a transcriptional regulatory sequence forcausing expression of said HDx polypeptide in eukaryotic cells; and (ii)a gene delivery composition for delivering said gene construct to a celland causing the cell to be transfected with said gene construct.
 11. Therecombinant transfection system of claim 10, wherein the gene deliverycomposition is selected from the group consisting of a recombinant viralparticle, a liposome, and a poly-cationic nucleic acid binding agent.